Novel peptide conjugates

ABSTRACT

Disclosed are a variety of peptide conjugates represented by the following general formula. 
     R 1 -Z-X-Z′-R 2   
     including methods of making and using such conjugates. Also provided are antibodies that specifically bind the peptide conjugates. The present invention has a wide spectrum of important applications including use in the treatment of disorders impacted by nociceptin and related opioid-like peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present provisional application is a continuation-in-part(CIP) of U.S. Provisional Application No. ______ entitled Novel PeptideConjugates filed on Jun. 13, 2001 by Larsen, B. D. et al. whichapplication is a CIP of U.S. Provisional Application No. 60/251,671filed on Dec. 6, 2000. The present application claims benefit to DanishPatent Applications DK PA2000 01485 filed on Oct. 5, 2000 and DK PA200000944 filed on Jun. 16, 2000. The disclosures of said U.S. ProvisionalApplication No. ______ filed on Jun. 13, 2001; No. 60/251,671; DK PA200001485; and DK PA2000 00944 applications are each incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to novel peptide conjugates havinginteresting pharmacological activities, a method of preparing the novelpeptide conjugates, pharmaceutical compositions containing the peptideconjugates, and the use of the peptide conjugates for the preparation ofa medicament.

BACKGROUND

[0003] The endogenous opioid-like peptide, nociceptin (also referred toas orphanin FQ), was first described in the central nervous system, andmost research in this field has focused on the CNS effects. Nociceptinbinds to a specific receptor named opioid receptor-like one (ORL1) withmuch greater affinity than to the three classical subtypes of opioidreceptors. Effects of nociceptin in the CNS include:hyperalgesia/hypoalgesia, stimulation of appetite and gnawing, increased(low doses) or decreased (high doses) locomotion, impaired learning, anddysphoria. However, nociceptin also exerts important effects outside theCNS. Thus, low doses of nociceptin increase the renal excretion of waterand decrease urinary sodium excretion (i.e., produces a selective waterdiuresis) which render this compound interesting for the treatment ofhyponatremia (Daniel R. Kapusta, Life Science, 60:15-21, 1997) (U.S.Pat. No. 5,840,696). When administered centrally (i.c.v.) or at highdoses peripherally (i.v. bolus or infusion), nociceptin decreases bloodpressure, heart rate and peripheral sympathetic nerve activity.

[0004] Dooley et al. (The Journal of Pharmacology and ExperimentalTherapeutics, 283(2):735-741, 1997) have shown that a positively chargedhexapeptide having the amino acid sequence Ac-RYY(RK)(WI)(RK)-NH₂, wherethe brackets show allowable variation of amino acid residue, acts as apartial agonist of the nociceptin receptor ORL1. Said hexapeptide wasidentified from a combinatorial peptide library and the sequence isunique without homology or similarity to the nociceptinheptadecapeptide. However, said hexapeptide is too unstable to yield asatisfactory medicament.

[0005] WO 99/44627 discloses the use of hexapeptides including thehexapeptides discovered by Dooley et al. for the manufacture of apharmaceutical composition for the treatment of the followingconditions: Migraine, type II diabetes, septic shock, inflammation andvasomotor disturbances. It is shown that the hexapeptideAc-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ inhibits depressor response to spinalcord stimulation.

[0006] Considering that ORL1 agonists including the hexapeptidediscovered by Dooley et al. as well as true nociceptin analogues areexpected to have serious CNS side effects, if exposed to the brain,which are observed at dose levels higher than those required to elicitwater diuresis in rats, it is a major objective of the present inventionto provide novel conjugated peptides having the nociceptin-like activityof said hexapeptide or binding activity at the ORL1 receptor, but actingselectively outside the CNS. It is a further object of the invention toprovide novel conjugated peptides having the nociceptin-like activity ofsaid hexapeptide or binding activity at the ORL1 receptor and havingimproved stability. Moreover, it is an object of the invention toprovide novel conjugated peptides having nociceptin-like activity orbinding activity at the ORL1 receptor and/or at a yet unidentifiedreceptor in kidney tissue.

[0007] This objective is achieved with the peptide conjugates of thepresent invention comprising hexapeptides modified C- and/orN-terminally by conjugation to short charged peptide chains.

SUMMARY OF THE INVENTION

[0008] A peptide conjugate of the general formula I

R₁-Z-X-Z′-R₂  (I)

[0009] wherein X represents a hexapeptide of the formula II

A¹-A²-A³-A⁴-A⁵-A⁶  (ii)

[0010] wherein

[0011] A¹ represents Arg, Lys, His or Asp, A² represents Tyr, Trp, orPhe, A³ represents Tyr, Asn,Trp or Phe, A⁴ represents Lys, Arg or His,A⁵ represents Phe, Tyr, Trp, Leu, Val or Ile, and A⁶ represents Arg, Lysor His and wherein each amino acid residue in said hexapeptide may be inthe L or D form;

[0012] Z represents a charged peptide chain of from 4 to 20 amino acidresidues having the D or L configuration or is missing; and Z′represents a charged peptide chain of from 4 to 20 amino acid residueshaving the D or L configuration or is missing, providing that not bothof Z and Z′ are missing;

[0013] R₁ represents H or an acyl group;

[0014] R₂ represents NR₃R₄ where each of R₃ and R₄ independentlyrepresents hydrogen, C(1-6)-alkoxy, aryloxy or a lower alkyl as definedherein; or R₂ represents OH; and

[0015] salts, hydrates and solvates thereof and C-terminally amidated oresterified derivatives thereof with suitable organic or inorganic acids.

[0016] The peptide conjugates of the invention can form acid additionsalts, preferably with pharmaceutically acceptable acids, and thesesalts are included within the scope of the invention. The compounds mayserve as medicaments in their pure form. However, the compounds arepreferably incorporated into either solid or liquid medical formulationsincluding tablets, capsules, solutions and suspensions. Other componentsof such formulations can include, a carrier, a diluent, a bufferingagent, a tonicity adjusting agent and a preservative. Solid formulationsare particularly suitable for oral administration, while solutions aremost useful for injection (i.v., i.m., i.p. or s.c.) or intranasaladministration.

[0017] The peptide conjugates of the invention having nociceptin-likeactivity or binding activity at the ORL1 receptor, but acting outsidethe CNS, are especially useful in the treatment of ORL1 relatedperipheral diseases and ailments, such as diseases where a selectiverenal effect is required or preferred. More specifically, the peptideconjugates of the invention exhibit a significant sodium-sparing andpotassium-sparing aquaretic effect, which is beneficial in the treatmentof edema-forming pathological conditions associated with hyponatremiaand/or hypokalemia. The peptide conjugates of the invention are usefulfor medical treatment of humans and for veterinarian use as a diureticduring edema-forming states in livestock, such as horses and cattle. Aparticular advantage of the compounds of the invention is theirstability in plasma compared to the hexapeptides disclosed by Dooley etal.^([1])

[0018] The novel peptide conjugates of the invention are useful in thepreparation of antibodies capable of specifically binding to saidpeptide conjugates. Antibodies raised against the peptide conjugates ofthe invention or fractions thereof are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows concentration-response curves of the effects ofcumulative addition of test substance (Ac-RYYRWK-NH₂ and Compound 1) onnociceptin-induced relaxation in the mouse vas deferens assay.

[0020]FIG. 2 shows cardiovascular and renal responses of i.v. infusednociceptin (11 nmol/kg/min); HR is heart rate (beats per minute, bpm),MAP is mean arterial pressure (mmHg), V is urine flow rate (μL/min), andUNaV is urinary excretion rate of sodium (μeq/min).

[0021]FIG. 3 shows cardiovascular and renal responses of i.v. infusedCompound 1 (1 and 0.1 nmol/kg/min) as well as renal sympathetic nerveactivity as percent of the control level (RNSA, %).

[0022]FIG. 4 shows the decrease in osmolar clearance (COsm) and theincrease in free water clearance (CH₂O) observed during i.v. infusion ofCompound 1 (1 and 0.1 nmol/kg/min).

[0023]FIG. 5 illustrates the similar magnitude of changes incardiovascular and renal responses (V and U_(Na)V) elicited by i.v.infusion of 1 nmol/kg/min of Compound 1 and 11 nmol/kg/min ofnociceptin.

[0024]FIG. 6 illustrates the similar magnitude of changes incardiovascular and renal responses (V and U_(Na)V) elicited by i.v.infusion of 0.1 nmol/kg/min Compound 1 and 1.1 nmol/kg/min ofnociceptin.

[0025]FIG. 7 is an illustration of the relationship between changes inurine flow rate, sodium and potassium excretion after i.v.administration of Compound 1, 65 nmol/kg i.v. (arrow) in normal rats.Data are expressed relative (percent) to the level prior to i.v.administration of Compound 1.

[0026]FIG. 8 illustrates changes in urine flow rate, free waterclearance, fractional potassium excretion, and fractional sodiumexcretion after i.v. administration of Compound 1, 65 nmol/kg i.v. innormal rats and cirrhotic rats.

[0027]FIG. 9 is an illustration of the fractional excretion of water(V/GFR) during steady-state i.v. infusion with a maximal aquaretic doseof the selective vasopressin type-2 receptor antagonist OPC-31260 (0.8mg/kg/h; n=8 (data from^([2])) or Compound 1 (12 nmol/kg/min). In orderto prevent activation of compensatory volume homeostatic mechanisms,experiments were performed during conditions whereby volume depletionwas prevented by computer-driven, servo-controlled intravenous volumereplacement with 150 mM glucose.

[0028]FIG. 10 is a graph showing the rate of degradation in heparinisedmouse plasma of Compound 1 and Ac-RYYRWK-NH₂.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Coupling of a charged peptide moiety introduces increasedpolarity to or increased charge of the positively charged hexapeptide(RK)YY(RK)(WI)(RK) resulting in a peptide conjugate with enhancedstability and hydrophilicity. Said coupling is also thought to decreasethe likelihood of the peptide conjugates crossing the blood-brainbarrier. Besides, preliminary data suggest that C-terminal modificationwith K₆ appears to induce the α-helix structure in the hexapeptide X asdetermined by 1D-NMR spectra. In preferred embodiments of the inventionformula II is represented by the amino acid sequence (RK)YY(RK)(WI)(RK)wherein alternative amino acid residues at positions 1, 4, 5 and 6 areshown in brackets. Alternatively, the amino acid residues R and K atpositions 1, 4 and 6 may each be substituted with Orn, Dab or Dapa. Inpreferred embodiments of the invention Z represents a negatively chargedpeptide chain of from 4 to 20 amino acid residues, Z′ represents apositively charged peptide chain of from 4 to 20 amino acid residues, R₁represents Ac or Tfa, and R₂ represents NH₂, or R₂ represents NR₃R₄where each of R₃ and R₄ independently represents hydrogen, methyl orethyl. Said hexapeptide X is preferably selected form the groupconsisting of KYYRWR, RYYRWR, KWRYYR, RYYRWK, RYYRWK (all-D), RYYRIK,RYYRIR, RYYKIK, RYYKIR, RYYKWR, and RYYKWK, more preferably the groupconsisting of RYYRWR, RYYRWK, RYYRIK, RYYKWR, and RYYKWK, morepreferably said hexapeptide X is RYYRWK or KYYRWK, wherein the aminoacid residues are in the L-form unless otherwise specified. Furthermore,the number of amino acid residues in each of Z and Z′ is preferably inthe range of 4-10 or 5-10.

[0030] It is preferred that the amino acid residues of Z are selectedfrom the group consisting of Q, T, S, P, N, E, and D having the D or Lconfiguration, and the N-terminal amino acid of Z is selected from thegroup consisting of Q T, N and S having the D or L configuration and theremaining amino acid residues are selected from the group consisting ofP, D and E. More specifically, Z is selected from the group consistingof, e.g. N(E)₇, N(E)₆, N(E)₅, N(E)₃, S(E)₇, S(E)₆, S(E)₅, S(E)₃, NP(E)₄,NP(E)₅, N(D)₇, N(D)₆, N(D)₅, N(D)₃, Q(E)₇, Q(E)₅, Q(E)₃, QN(D)₇, Q(D)₆,Q(D)₅, and Q(D)₃, preferably Z is N(E)₅. It is preferred that the aminoacid residues of Z′ are selected from the group consisting of A, G, K,and R, preferably K, having the D or L configuration, more preferably Z′is selected from the group consisting of A(K₄)G, K₅G, AK₅, H₆, K₁₀, K₈,K₆, K₅, and K₄. Examples of conjugates Z-X-Z′ are N(E)₅RYYRWKK₆,N(E)₅RYYRWK, RYYRWKK₆, N(E)₅RYYRWRK₆, N(E)₅RYYRWR, RYYRWRK₆,N(E)₅RYYRIKK₆, N(E)₅RYYRIK, and RYYRIKK₆. Exemplary compounds of theinvention are: Ac-RYYRWKK₆-NH₂, Ac-K₆RYYRWK-NH₂, N(E)₅RYYRWKK₆-NH₂, andsalts, preferably pharmaceutically acceptable salts, hydrates, solvatesand derivatives thereof including C-terminal derivatives, such as freecarboxylic acid.

[0031] A further preferred embodiment of the invention is represented bythe general formula III

R₁-X-Z′-R₂  (III)

[0032] wherein R₁, X, Z′ and R₂ have the same meanings as defined above,and salts, hydrates and solvates thereof and C-terminally amidated oresterified derivatives thereof with suitable organic or inorganic acids.

[0033] In a preferred peptide conjugate of formula III R₁ represents Ac,X represents a hexapeptide of the formula (RK)YY(RK)(WI)(RK) whereinalternative amino acid residues at positions 1, 4, 5 and 6 are shown inbrackets, Z′ represents K_(n) where n is an integer selected from 5, 6or 7, and R₂ represents NH₂. X is preferably selected from the groupconsisting of KYYRWK, RYYRWR, RYYRWK, RYYRWK (all-D), KWRYYR, RYYRIK,RYYKWR, and RYYKWK. The peptide conjugates of formulae I and III areoptionally further linked to a transport moiety or an affinity tag, suchas H₆, where the linkage between the peptide conjugate and saidtransport moiety or affinity tag may be by any convenient covalent bond.Said transport moiety is preferably selected from the group consistingof a HIV tat peptide residues 49-57, HIV tat peptide residues 49-56, thetat sequence YGRKKRRQRRR, a polyarginine peptide having from 6 to 20residues, such as R₆, and transducing peptide sequences, such as thefollowing peptide sequences:

[0034] YARKARRQARR, YARAAARQARA, YARMRRMRR, YARAARRMRA, ARRRRRRRRR, andYAAARRRRRRR, which are disclosed in WO 99/29721 and in U.S. Pat. No.6,221,355 (seq. id. nos. 3-8) the disclosures of which are incorporatedby reference.

[0035] The peptide sequence of a compound of formulae I and III isoptionally in the all-D form, the retro form or the retro all-D form,where the all-D form is more preferred. Optionally, the X sequence offormulae I and III is an all-D form, a retro form, or a retro all-D formof the peptide sequence of formula II or the hexapeptide(RK)YY(RK)(WI)(RK) as defined above, respectively.

[0036] Exemplary compounds of the invention are shown in Table 1 below:Compound 1 Ac-RYYRWKKKKKKK-NH₂ Compound 2 Ac-KKKKKKRYYRWK-NH₂ Compound 3H-NEEEEERYYRWKKKKKKK-NH₂ Compound 4 Ac-RYNRWKKKKKKK-NH₂ Compound 5Ac-KKKKKKKWRYYN-NH₂ Compound 6 Ac-KKKKKKKWRYYR-NH₂ Compound 7Ac-KKKKKKKWRYYR-NH₂ (all D) Compound 8 Ac-RYYRWKKKKKKK-NH₂ (all D)Compound 9 Ac-KYYRWKKKKKKK-NH₂ Compound 10 Ac-RYYRIKKKKKKK-NH₂ Compound11 Ac-RYYRWKAKKKKK-NH₂ Compound 12 Ac-RYYRWKKKKKK-NH₂ Compound 13Ac-RYYRWKKKKKKKC-NH₂ Compound 14 Tfa-RYYRWKKKKKKK-NH₂

[0037] and salts, hydrates, solvates and C-terminal derivatives thereof,such as the free carboxylic acid. Other preferred compounds are

[0038] Ac-KWRYYNKKKKKK-NH₂

[0039] Ac-KWRYYRKKKKKK-NH₂ and

[0040] Ac-KWRYYRKKKKKK-NH₂ (all D) and salts, hydrates, solvates andC-terminal derivatives thereof, such as the free carboxylic acid.

[0041] Examples of acid addition salts with an organic and an inorganicacid of compounds of the invention are

[0042] Compound 1A Ac-RYYRWKKKKKKK-NH₂×9CH₃COOH and Compound 1CAc-RYYRWKKKKKKK-NH₂×9HCl.

[0043] The peptide conjugates of the invention are preferably preparedusing the method of synthesis disclosed in WO 98/11125 the disclosure ofwhich is incorporated by reference, and preferably using a method asdescribed in Example 15 therein. Said methods of synthesis will resultin a primary peptide product having a trifluoroacetate counterion andwhich may be suitable for the preparation of a medicament. In manyinstances, however, it may be advantageous to perform a counter ionexchange from trifluoroacetate to a pharmaceutically acceptable orpreferred anion, such as acetate. This may be effected by ion exchangechromatography. Alternatively, the primary peptide product may berepeatedly freeze dried and dissolved in diluted hydrochloric acid toobtain the purified hydrochloride.

[0044] Also included in the present invention is the use of a peptideconjugate of formula I or III or an epitopic fragment thereof comprisinga part or all of the X sequence of formula II or the formula(RK)YY(RK)(WI)(RK) and/or a part or all of the Z sequence or the Z′sequence preferably coupled to a carrier through a terminal cysteinylresidue for raising antibodies capable of specifically binding to saidpeptide conjugates or said X sequence. The terminal Cys residue isoptionally obtained through the substitution with a Cys residue of oneof the terminal aminoacid residues of said peptide conjugate or saidepitopic fragment thereof, or said peptide conjugate or said epitopicfragment thereof comprises a further Cys residue at one of its termini.Examples of peptide fragments having a terminal C useful for raisingantibodies capable of specifically binding to the peptide conjugates ofthe invention are RYYRWKC, KYYRWKC, CWKKKKKKK, CKKKKKK, and CWKKKKKK. Inaddition, peptide fragments having a considerable sequence homology tothe peptide conjugates of the invention can be used for raisingantibodies capable of specifically binding to the peptide conjugates ofthe invention. Examples are CAPPSKKKKKK, CMPKKKKKK, CPPSKKKKKK, etc.Optionally, each of the foregoing peptide conjugates having a Cysterminus can have at least one amino acid in the D form.

[0045] As will be discussed in more detail below, the novel peptideconjugates can be administered as a sole active agent or in combinationwith one or more medicaments such as those specifically provided below.

[0046] Throughout the description and claims the one letter code fornatural amino acids is used as well as the three letter code for naturalamino acids and generally accepted three letter codes for other (α-aminoacids, such as Ornithine (Orn), 2,4-Diaminobutanoic acid (Dab) and2,3-Diaminopropanoic acid (Dapa). Where the L or D form has not beenspecified it is to be understood that the amino acid in question has thenatural L form, cf. Pure & Appl. Chem. Vol. 56(5) pp595-624 (1984).Where nothing is specified it is to be understood that the C-terminalamino acid of a compound of the invention exists as the free carboxylicacid, this may also be specified as “—OH”. Cf. Biochem. J., 1984, 219,345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2,Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem.,1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids andPeptides, 1985, 16, 387-410; Biochemical Nomenclature and RelatedDocuments, 2nd edition, Portland Press, 1992, pages 39-69; CopyrightIUBMB and IUPAC. The term “retro form” of a peptide conjugate offormulae I or III refers to a peptide having the reversed aminoacidsequence of formulae I or III. The term “all-D form” of a peptideconjugate of formulae I or III refers to a peptide wherein all aminoacidunits are in the D-form. The term “retro all-D form” of a peptideconjugate of formulae I or III refers to a peptide having the reversedaminoacid sequence of formulae I or III and wherein all aminoacid unitsare in the D-form (retro-inverse form). Optionally, in a peptideconjugate of the invention the sequence defined by formula II or theformula (RK)YY(RK)(WI)(RK) is in the all-D form, the retro form or theretro all-D form. D-aminoacids are unnatural aminoacids which are stablein a protease rich environment. Thus, a useful way of stabilising thepeptide conjugates of the invention against proteolytic degradation isto substitute L-aminoacids with corresponding D-aminoacids. The term“epitopic fragment” refers to a truncated or shortened section of apeptide sequence having, e.g., 5-8 aminoacid units and is capable ofeliciting an antigen response.

[0047] The term “alkyl” refers to univalent groups derived from alkanesby removal of a hydrogen atom from any carbon atom: C_(n)H_(2n+1)—. Thegroups derived by removal of a hydrogen atom from a terminal carbon atomof unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups:H[CH₂]_(n)−. The groups RCH₂—, R₂CH—(R not equal to H), and R₃C—(R notequal to H) are primary, secondary and tertiary alkyl groupsrespectively. “Alkyl” refers to any alkyl group, and includesC(1-6)alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, pentyl andhexyl and all possible isomers thereof. By “lower alkyl” is meantC(1-6)alkyl, preferably C(1-4)alkyl, more preferably, methyl and ethyl.

[0048] The term “C(1-6)alkoxy” refers to an ester group of the formulaR—O— wherein R represents C(1-6)alkyl. The term “aryloxy” refers to anester group of the formula R—O— wherein R represents phenyl or naphthyloptionally substituted with a lower alkyl group.

[0049] The term “acyl” as used herein include acyl radicals which areformally derived from oxoacids R_(k)E(═O)_(l)(OH)_(m)(I not equal to 0)by removal of a hydroxyl cation HO⁺, a hydroxyl radical HO⁻or a hydroxylanion HO⁻, respectively, and replacement analogues of suchintermediates. Acyl radicals can formally be represented by canonicalforms having an unpaired electron or a positive charge on theacid-generating element of the oxoacid. Acyl radicals, e.g. RC(═O),RS(═O)₂.

[0050] The term “acylated” as used herein indicates the the compound inquestion carries an acyl group. An acyl group is formed by removing oneor more hydroxy groups from an oxoacid, such as a carboxylic acid, thathas the general structure R_(k)E(═O)_(l)(OH)_(m) (I not equal to 0), andreplacement analogues of such acyl groups. E.g.CH₃C(═O)—, CH₃C(═NR)—,CH₃C(═S)—, PhS(═O)₂—, HP(≡N)—. In organic chemistry an unspecified acylgroup is commonly a carboxylic acyl group. Cf. International Union ofPure and Applied Chemistry, Recommendations on Organic & BiochemicalNomenclature, Symbols & Terminology etc. IUPAC Recommendations 1994.“Ac” indicates an acetyl group and “Tfa” indicates a trifluoroacetylgroup.

[0051] The term “peptide conjugate” as used herein indicates a fusionbetween at least two peptide sequences via a peptidic bond or anequivalent bioisosteric bond, such as the peptide bond mimeticsdescribed in Table 1 in Tayar et al., Amino Acids (1995) 8:125-139.

[0052] The term “transport moiety” as used herein indicates a chemicalentity that acts in escorting molecules such as polypeptides acrossbiological membranes. The transport polymers disclosed in WO 98/52612are all incorporated by reference. Peptide conjugates of the inventionare linked via a covalent bond to said transport moiety. The term “HIVtat peptide residues 49-57” refers to a transport moiety having thesequence RKKRRQRRR as disclosed in WO 91/09958.

[0053] “Agonist” refers to an endogenous substance or a drug that caninteract with a receptor and initiate a physiological or apharmacological response characteristic of that receptor (contraction,relaxation, secretion, enzyme activation, etc.).

[0054] “Antagonist” refers to a drug or a compound that opposes thephysiological effects of another. At the receptor level, it is achemical entity that opposes the receptor-associated responses normallyinduced by another bioactive agent.

[0055] “Partial agonist” refers to an agonist which is unable to inducemaximal activation of a receptor population, regardless of the amount ofdrug applied (See also Intrinsic activity). A “partial agonist” may alsobe termed “agonist with intermediate intrinsic efficacy” in a giventissue. Moreover, a partial agonist may antagonize the effect of a fullagonist that acts on the same receptor.

[0056] “Receptor” refers to a molecule or a polymeric structure in or ona cell that specifically recognizes and binds a compound acting as amolecular messenger (neurotransmitter, hormone, lymphokine, lectin,drug, etc.).

[0057] The term “salt”, as used herein, denotes acidic and/or basicsalts, formed with inorganic or organic acids and/or bases, preferablybasic salts. While pharmaceutically acceptable salts are preferred,particularly when employing the compounds of the invention asmedicaments, other salts find utility, for example, in processing thesecompounds, or where non-medicament-type uses are contemplated. Salts ofthese compounds may be prepared by art-recognized techniques. Examplesof such pharmaceutically acceptable salts include, but are not limitedto, inorganic and organic acid addition salts, such as hydrochloride,sulphates, nitrates or phosphates and acetates, trifluoroacetates,propionates, succinates, benzoates, citrates, tartrates, fumarates,maleates, methane-sulfonates, isothionates, theophylline acetates,salicylates, respectively, or the like. Lower alkyl quaternary ammoniumsalts and the like are suitable, as well. “Pharmaceutically acceptableanions” as used herein includes the group consisting of CH₃COO⁻,CF₃COO⁻, Cl⁻, SO₃ ²⁻, maleate and oleate.

[0058] The term “peripheral administration” includes all administrationforms that exclude delivery of the active substance directly into thecentral nervous system. “Central administration” as used herein means anadministration directly into the central nervous system, such asintracerebroventricular administration (i.c.v. administration).

[0059] The term “hyponatremia” as used herein includes but is notnecessarily limited to the following medical conditions:

[0060] Pseudohyponatremia characterised by Normal plasma osmolalityassociated with, e.g., hyperlipidemia, hyperproteinemia orposttransurethral resection of prostate/bladder tumor, and

[0061] Increased plasma osmolality associated with, e.g., hyperglycemiaand mannitol;

[0062] Hypoosmolal hyponatremia characterised by Primary Na⁺loss(secondary water gain) associated with, e.g., integumentary loss:sweating, burns; gastrointestinal loss: vomiting, tube drainage,fistula, obstruction, diarrhea; renal loss: diuretics, osmotic diuresis,hypoaldosteronism, salt-wasting nephropathy, postobstructive diuresis,nonoligouric acute tubular necrosis;

[0063] Primary water gain (secondary Na⁺loss) associated with, e.g.primary polydipsia; decreased solute intake (e.g., beer potomania); AVP(vasopressin) release due to pain, nausea, drugs; syndrome ofinappropriate AVP secretion; glucocorticoid deficiency; hypothyroidismand chronic renal insufficiency; and

[0064] Primary Na⁺gain (exceeded by secondary water gain) associatedwith, e.g. heart failure, hepatic cirrhosis and nephrotic syndrome.

[0065] Congestive Heart Failure (CHF)

[0066] Congestive heart failure (CHF) is the pathophysiological state inwhich an abnormality in cardiac function is responsible for the failureof the heart to pump blood at a rate commensurate with the requirementsof the metabolizing tissues. Regardless of the underlying cause of CHF(e.g., ischemic heart disease, arterial hypertension, dilatedcardiomyopathy, valvular disease, anemia etc.), the major clinicalmanifestation of heart failure is dyspnea which appears with lessstrenuous activity as the disease progresses. According to the New YorkHeart Association (NYHA) functional classification, the CHF patients maybe classified into one of four classes (class I, class II, class III,class IV). This functional classification is used worldwide and has astrong association with mortality, which is independent of leftventricular ejection fraction.

[0067] Congestive heart failure is characterized by a complex series ofneurohumoral adjustments. The major mechanisms are activation of thesympathetic nervous system, stimulation of therenin-angiotensin-aldosterone axis, and stimulation of vasopressinrelease. These influences elevate systemic vascular resistance andenhance sodium and water retention, while potassium excretion isincreased due to hyperaldosteronism. Due to activation of waterretaining mechanisms patients with end-stage CHF have a reduced capacityto excrete water, and unless water intake is restricted, these patientsare at great risk of developing dilutional hyponatremia.

[0068] Current Therapy of CHF

[0069] The purpose of medical treatment is to relieve the patients fromdebilitating symptoms without compromising the myocardial function tooseverely. During progression of CHF, the medical treatment isintensified by addition of drugs with different sites of action. Thecurrent treatment of heart failure can be divided into three categories:(1) Reduction of cardiac workload including preload and afterload; (2)control of excessive retention of water and salt; (3) enhancement ofmyocardial contractility. NYHA class IV patients are usually intreatment with maximal doses of vasodilators, such as ACE inhibitors,hydralazine, alpha-adrenergic inhibitors, and nitrates. However,pulmonary congestion due to water and salt retention is still a majorproblem for these patients. Despite the intensive medical therapy withdrugs from all classes, the mortality and morbidity is still very highin patients with CHF.

[0070] Patients in severe heart failure (NYHA class III-IV) usuallyrequire a combination of at least two different diuretics with differenttubular sites of action. The most commonly used is a combination of aloop diuretic (e.g., furosemide, bumetanide) and a thiazide diuretic(e.g., chlorothiazide, bendroflumethiazide). High doses of thiscombination are the most effective therapy for producing natriuresis(maximal efficacy is about 30% of filtered load for loop diuretics andabout 10% for thiazides). However, blockade of the thiazide-sensitivesodium reabsorption in the distal convoluted tubules (i.e., the dilutingsegment) may produce hypertonic urine and contribute to dilutionalhyponatremia. Hyperaldosteronism and the associated increase in deliveryof sodium to the collecting ducts, stimulates sodium-potassium exchangein the collecting ducts resulting in kaliuresis. Thus, combinationtherapy with loop diuretics and thiazides is the most common cause forboth hyponatremia and hypokalemia in heart failure patients.Potassium-sparing diuretics (e.g., spironolactone, amiloride) may beused in combination with thiazides or loop diuretics, but the poormaximal efficacy (max. 3% of filtered water) of these compounds makesthese drugs weak diuretics.

[0071] In addition, due to hyperaldosteronism, patients with end-stageCHF are at great risk of developing hypokalemia.

[0072] When symptoms such as dyspnea at rest, orthopnea and paroxysmalnocturnal dyspnea persist despite maximal medical treatment with loopdiuretics (=loop-diuretic resistant CHF or refractory CHF), additionaldiuretic therapy is necessary to avoid life threatening pulmonarycongestion. Thiazides are often added as the first drug of choice whenthe therapeutic effect of loop diuretics fails. The combination of loopdiuretics+ thiazides produce an effective natriuresis, however,inhibition of sodium reabsorption in the thiazide-sensitive dilutingsegment of the nephron, often results in hypertonic urine, which mayresult in serious electrolyte disturbances. Thus, combination of loopdiuretics and thiazides is the most common cause of hypokalemia andhyponatriemia in patients with NYHA class IV heart failure^([3]).

[0073] Hypokalemia is a major predisposing mechanism for the developmentof arrhythmias and the prognosis of CHF is poor when serum potassiumlevels fall below 3.3 mM. Furthermore, in CHF patients treated withdigoxin, hypokalemia is the most common precipitating cause of digitalisintoxication, which is a serious and potentially fatal complication.

[0074] In patients with severe heart failure, combination treatment withloop diuretics and thiazides often produces hypotonic hyponatremia as aconsequence of increased sodium losses and impaired water excretion. Ifthe hyponatremia develops faster than the brain can adapt, symptoms ofcerebral edema develop (e.g., headache, nausea, vomiting, weakness,incoordination, tremors, delirium, and ultimately seizures and coma).Deaths occur when the serum level of sodium (S—Na) falls below 120 mM ata rate that exceeds 1 mmol/l/hr. When the hyponatremia develops moregradually, the symptoms are more subtle, vague and non-specific and theytend to occur at even lower S—Na levels. Typically symptoms includepersonality changes (depression, non-cooperation, confusion), anorexia,nausea, muscular weakness, and cramps. When S—Na<120 mM gaitdisturbances, stupor, and seizures may occur.

[0075] Class IV represents the group of patients with end-stage heartfailure. Among the characteristic clinical manifestations, the dyspneaat rest is the most striking symptom. As heart failure advances dyspneaappears with progressively less strenuous activity, however, duringend-stage heart failure, dyspnea even in the recumbent position(orthopnea) is a common finding. The patients have engorged pulmonaryvessels and interstitial pulmonary edema, which is often evident onradiological examination. Patients with orthopnea must elevate theirheads to relieve the pulmonary congestion, and they are frequently awakeduring night with severe attacks of coughing and the sensation ofbreathlessness (nocturnal dyspnea). This condition is quite frighteningand the anxiety often worsens the symptoms of heart failure.

[0076] Along with pulmonary congestion in class IV patients, the reducedcardiac function is associated with congestion of the liver and theportal venous system, which produces anorexia, nausea and abdominalpain. Patients belonging to class IV have an extremely high 1-yearmortality (30-80%). The major causes of death are sudden death (40%) andworsening of the congestive heart failure (40%). Among the factors thatare associated with poor prognosis is low ejection fraction (<25%),reduced oxygen uptake, reduced serum sodium concentration (<133 mM), andreduced serum potassium concentration (<3 mM)^([3]). The peptideconjugates of the invention exhibit an advantageous pharmacologicalprofile suggesting an ability to alleviate pulmonary congestion,decrease pre- and afterload, increase cardiac output and oxygen uptake,and prevent hyponatremia and hypokalemia.

[0077] When cardiac output is reduced a series of compensatoryadjustments occurs that ultimately results in the abnormal accumulationof fluid. In CHF patients with NYHA Class I through III, the abnormalfluid accumulation and the accompanying expansion of blood volumeconstitutes an important compensatory mechanism that tends to maintaincardiac output and perfusion to vital organs. However, in the terminalstage of heart failure (NYHA class IV) the cardiac ventricles operate ona depressed and flattened function curve, and in terminal heart failure,the patient end up on the descending part of the Starling volume-cardiacoutput curve. Thus, these patients need effective diuretic therapy inorder to control blood volume and prevent life threatening pulmonaryedema. The advantageous effects of the peptide conjugates of theinvention in heart failure, such as loop diuretic-resistant refractoryheart failure, are to decrease blood volume and pulmonary congestion. Ifthe patient is on the flattened part of the curve, cardiac outputdecreases slightly, and if the patient is on the descending part of thecurve cardiac output may increases^([3]).

[0078] Having a unique pharmacological profile as a potassium- andsodium-sparing aquaretic, the peptide conjugates of the invention, suchas Compound 1, represent the ideal candidate drugs for the treatment ofCHF symptoms in patients with refractory heart failure who no longerrespond sufficiently to high doses of loop diuretics. In these patients,the novel peptide conjugates as an add-on or combination therapy to loopdiuretics may promote an effective diuresis without the acceleratedsodium and potassium losses seen with other diuretics. The same uniquepharmacological profile will also present an advantage in the treatmentof hyponatremia, renal disease, and edema associated with nephroticsyndrome and liver cirrhosis.

[0079] Pharmacology

[0080] Mouse Vas Deferens Assay

[0081] The compounds of the present invention are pharmacologicallyactive, e.g. as nociceptin agonists or partial agonists, and a usefulmethod of showing activity is described by Hughes J., Kosterlitz H. W.,Leslie F. M.: Effect of morphine on adrenergic transmission in the mousevas deferens. Assessment of agonist and antagonist potencies of narcoticanalgesics^([4]). The isolated electrically stimulated mouse vasdeferens preparation has been widely used to evaluate the functionalpharmacological actions of opioid agonists and antagonists invitro^([5]). The mouse vas deferens possesses all three opioid receptortypes and subsequent studies have described that the the mouse vasdeferens assay is also sensitive to nociceptin^([6]).

[0082] The vas deferens from male NMRI albino mice weighing 30-40 g wereplaced in a SCHULER organ bath Type 809, Hugo Sachs Elektronik, Germany,and perfused with a 37° C., modified Krebs solution (NaCl 6,9 g/L, KCl0.35 g/L, KH₂PO₄0.16 g/L, NaHCO₃2.1 g/L, Glucose H₂O 2.0 g/L, CaCl₂2.52mL/L). was gassed with carbogen (95% O₂ and 5% CO₂) throughout theexperiment. The tissue was electrically stimulated through two silverelectrodes directly in the organ bath using a HSE Stimulator I Type 215.Stimulation of the tissue was performed by applying a supramaximalvoltage (15 Volts) in trains of 10 square pulses every 60 s, each of 1ms duration with a frequency of 100 ms. Concentration-response curveswere performed by adding test substances that relax mouse vas deferenssmooth muscles cumulatively to the organ baths. The test substances usedin the assay were nociceptin in an isotonic saline solution, Compound 1(as the trifluoroacetate in an isotonic saline solution), Compound 2 (asthe trifluoroacetate in an isotonic saline solution), and thehexapeptide Ac-RYYRWK-NH₂ (as the trifluoroacetate in an isotonic salinesolution). The peptide conjugates of the present invention arebiologically active in the mouse vas deferens (MVD) assay, but thecompounds inhibit electrically-induced contraction of the mouse vasdeferens with varying potency: nociceptin>Compound 1=Compound2>>Ac-RYYRWK-NH₂(data not shown).

[0083] The effects of Ac-RYYRWK-NH₂ and Compound 1 on nociceptin-inducedrelaxation in the mouse vas deferens assay were also tested. Resultsfrom these experiments are shown in FIG. 1. These data demonstrate thatboth the partial agonist Ac-RYYRWK-NH₂ ^([1]) and the peptide conjugatereferred to as Compound 1 inhibit nociceptin-induced smooth musclerelaxation. Using the Schild equation^([7]), the pA₂ values whichindicates affinity of a compound as an antagonist were calculated. Theseresults are presented in Table 2: TABLE 2 Ac-RYYRWK-NH₂ Compound 1 pA₂value (number of 7.8 (n = 4) 9.5 (n = 4) experiments)

[0084] These data demonstrate that Compound 1 is about 50-fold morepotent as an antagonist on the nociceptin-induced smooth musclerelaxation in the MVD assay than the unconjugated hexapeptideAc-RYYRWK-NH₂.

[0085] In Vivo Studies

[0086] Previous studies have demonstrated that nociceptin produces amarked increase in urine flow rate and decrease in urinary sodiumexcretion (i.e., an aquaretic response) when administered centrally(intracerebro-ventricularly, i.c.v.) or as an i.v. infusion in consciousrats^([8-10]). In the conscious rat model, the animal is chronicallyinstrumented with catheters in the urinary bladder, the femoral artery,and the femoral vein. The animal receives a continuous i.v. infusionwith isotonic saline, 50 μl/min. Urine is collected in vials inconsequtive urine collection periods of 10 min each. After two controlperiods, the test compound is administered and fractionated urinecollections (10 min periods) are continued for at least two hours.Urinary concentrations of sodium and potassium are determined by flamephotometry (Instrumentation Laboratory 943) using caesium as internalstandard.

[0087] The diuretic effect of the peptide conjugates of the inventionhas been tested in conscious rats, where Compound 1 elicits a powerfuland sustained diuretic response. Cardiovascular and renal responsesproduced by i.v. infusion of nociceptin (Phoenix Pharmaceuticals) andCompound 1 have been studied in conscious Sprague Dawley rats. Theresults are shown in FIGS. 2 to 8 wherein HR is heart rate, MAP is meanarterial pressure, V is urine flow rate, U_(Na)V is urinary excretionrate of sodium, RSNA is renal sympathetic nerve activity expressed as %of the control level, C_(osm) is the osmolar clearance, and C_(H2O) isfree water clearance.

[0088]FIG. 2 shows the typical cardiovascular and renal responses ofi.v. infused nociceptin (11 nmol/kg/min). Nociceptin significantlydecreases MAP possibly due to relaxation of arterial smoothmuscle^([11]).

[0089]FIG. 3 shows significant diuretic (V μl/min) and antinatriureticresponses (U_(Na)V μeq/min) of i.v. infused Compound 1 (1 and 0.1nmol/kg/min). Similar to i.v. nociceptin, Compound 1 produced a slight,but significant reduction in MAP with little effect on HR. All ratsinfused with compound 1 showed no signs of increased appetite, sedation,or behavioral changes normally associated with nociceptin treatment.

[0090]FIG. 4 shows the decrease in osmolar clearance and the increase infree water clearance observed during i.v. infusion of Compound 1 (1 and0.1 nmol/kg/min).

[0091]FIG. 5 illustrates the similar magnitude of changes in V andU_(Na)V elicited by i.v. infusion of 1 nmol/kg/min Compound 1 and 11nmol/kg/min of nociceptin.

[0092]FIG. 6 illustrates the similar magnitude of changes in V andU_(Na)V elicited by i.v. infusion of 0.1 nmol/kg/min Compound 1 and 1.1nmol/kg/min of nociceptin. Together the data presented in FIGS. 5 and 6suggest that Compound 1 compared to authentic nociceptin isapproximately 10-fold more potent in producing similar magnitudediuretic and antinatriuretic responses. The diuretic effect of Compound1 infusion tended to persist for a greater duration than that of i.v.nociceptin infusion.

[0093] Furthermore, the effects of i.v. bolus administration ofnociceptin (30 and 100 nmol/kg, Phoenix Pharmaceuticals) andnociceptin-NH₂ (30 nmol/kg) on heart rate, arterial pressure and RSNAwere studied in groups of 6 conscious Sprague-Dawley rats. The bolusinjection produced rapid bradycardia, a hypotensive response and initialRSNA suppression (data not shown). A similar i.v. bolus injection ofCompound 1 (30 nmol/kg) did not produce any changes in heart rate, bloodpressure or RSNA. In addition, rats tested at this dose do not show anysigns of stimulation of appetite or behavioral changes. The i.v. bolusinjection of Compound 1 (30 nmol/kg) in this same rat produced a markeddiuretic (V) and antinatriuretic (UNaV) response (data not shown). Asummary of in vivo responses to Compound 1 obtained in the experimentsdescribed above are shown in Table 3 below: TABLE 3 In vivo responsesfrom series of experiments where I.v. dose of Food Behavioral Compound 1HR MAP V U_(Na)V C_(H2O) intake changes I.v. infusion 0 (↓) ↑ 0 ↑ 0 00.1 nmol/kg/min I.v. infusion 0 (↓) ↑↑ ↓ ↑↑ 0 0 1.0. nmol/kg/min I.v.bolus injection 0 0 ↑ ↓ ↑ 0 0 30 nmol/kg

[0094] From these studies it appears that Compound 1 evokes asignificant diuretic response at i.v. infusion doses well below thatrequired to evoke feeding or behavioral changes (e.g., sedation,increased exploratory behavior, catalepsy, convulsion etc.).Furthermore, after 120 min of i.v. infusion of Compound 1 (1nmol/kg/min) in the study presented in FIG. 3, the HR and MAP responsesto an i.v. bolus injection of nociceptin were examined. In thesestudies, the typical bradycardia and hypotension (rapid in onset andlarge in magnitude) was completely blocked in all rats tested (n=6).

[0095] In an additional series of dose-response studies, thecardiovascular and renal responses to Compound 1 were investigated aftereither i.v. bolus injection (10 (n=6), 30 (n=4), 100 (n=6) or 300 (n=4)nmol/kg i.v.) or during continuous i.v. infusion (0.1 (n=6), 1 (n=6) or10 nmol/kg/min (n=6)). After stabilization of urine flow rate andurinary sodium excretion, urine was collected during a 20-min controlperiod. After this, the i.v. bolus injection was given or the i.v.infusate was switched to a solution of isotonic saline containingCompound 1 (i.v. infusion experiments). From the time of drugadministration, experimental urine samples (10-min consecutive periods)were taken for 80 min.

[0096] Feeding Response Observations

[0097] One hour prior to the start of each experiment a food pellet(standard rodent chow) was placed within access to the rat though a holein the rat holder. The number and time of feeding responses (i.e., timesin which the rat began to eat the food pellet) was recorded before, andafter drug administration. If a positive feeding response was observed,the pellet was removed from the restrainer to prevent feeding-inducedalterations in heart rate and/or mean arterial pressure. At 10-minintervals, the food pellet was presented to the rat to determine theduration of the feeding response.

[0098] In this series of studies in conscious, chronically catheterizedrats, we have demonstrated that i.v. bolus injection of Compound 1produces a selective water diuresis. Data are presented in Table 4below: TABLE 4 Responses after i.v. bolus administration of Compound 1Parameters I.v. bolus dose (nmol/kg) studied 10 (n = 6) 30 (n = 4) 100(n = 6) 300 (n = 4) Δ Urine Flow (%) +83 ± 42   +207 ± 11    +249 ±60    +127 ± 47    Δ Na −44 ± 3    −51 ± 7    −70 ± 2    −85 ± 6   excretion (%) Δ K −35 ± 4    −20 ± 7    −52 ± 5    −79 ± 6    excretion(%) Δ Blood −4 ± 1   −8 ± 2   −12 ± 2    −15 ± 3    Pressure (%) Foodintake − (+)* (+)** (+)***

[0099] When administered as a continuous i.v. infusion, a selectivewater diuretic response (diuresis associated with antinatriuresis) wasobserved at 1 and 10 nmol/kg/min (Table 5). TABLE 5 Responses duringi.v. infusion of Compound 1 I.v. infusion dose (nmol/kg/min) Parametersstudied 0.1 (n = 6) 1.0 (n = 6) 10 (n = 6) Δ Urine Flow (%) +99 ± 21  210 ± 51  160 ± 68  Δ Na excretion (%) −5 ± 3   −64 ± 7    −79 ± 6    ΔK excretion (%) −16 ± 6    −51 ± 9    −68 ± 15   Δ Blood Pressure (%) −5± 4   −10 ± 2    −15 ± 3   Food intake − − (+)*

[0100] Collectively, these dose-response studies demonstrate thatCompound 1 produces a significant water diuretic response at i.v.infusion and i.v. bolus injection doses that are well below thoserequired to elicit marked changes in cardiovascular function. This is incontrast to the diuresis produced by endogenous nociceptin, which isproceeded by a marked bradycardic and hypotensive response (i.v. bolusand i c.v. injection), and a concurrent inhibition of renal sympatheticnerve activity (i.c.v. injection),^([10]).These observations suggestthat unlike the endogenous ORL1 ligand, nociceptin, Compound 1 hasclinical utility as an aquaretic in patients at therapeutic dosessubstantially below those required to evoke adverse systemiccardiovascular events. We speculate that less penetration of Compound 1into the CNS may explain the more selective renal effect of the novelcompound compared to endogenous nociceptin.

[0101] Rats with liver cirrhosis induced by common bile duct ligationdevelop avid sodium and water retention and ascites within 2-4 weeks.The disturbed sodium and water balance in cirrhotic rats is in agreementwith the pivotal clinical findings of edema and ascites in patients withliver cirrhosis. To examine the efficacy of Compound 1 in anexperimental model of an edema-forming state, the diuretic response toan acute i.v. bolus injection of Compound 1 (65 nmol/kg) was evaluatedin rats with liver cirrhosis induced by common bile duct ligation 4weeks earlier. Prior to the experiments, rats were instrumented withchronic catheters in the urinary bladder, and in the femoral vein andartery. Renal responses to Compound 1 were compared with responses insham-operated control rats. Compound 1 produced a marked increase infree water clearance (water diuresis), associated with a decrease inurinary excretion of sodium and potassium in the absence of changes inthe glomerular filtration rate in all animals. The renal responses weresimilar in cirrhotic and control rats. Relative changes in urine flowrate, sodium and potassium excretion after i.v. administration of 65nmol/kg in sham-operated control animals are summarized in FIG. 7.

[0102] As illustrated in FIG. 8, cirrhotic rats had a significantlylower fractional sodium excretion prior to drug administration relativeto control animals. This indicates that the cirrhotic rats had severesodium retention, which is in accordance with the marked sodiumretention that characterizes human liver cirrhosis. Compound 1 produceda marked increase in urine flow rate, and free water clearance (waterdiuresis), associated with a decrease in fractional excretion of sodiumand potassium in the absence of changes in the glomerular filtrationrate in all animals. The renal responses were similar in cirrhotic andcontrol rats. Changes in urine flow rate, free water clearance,fractional sodium excretion and fractional potassium excretion afteri.v. administration of 65 nmol/kg in sham-operated control and cirrhoticanimals are summarized in FIG. 8. These results in an animal model of apathological state characterized by avid sodium retention indicate thatcompounds of the present invention, such as Compound 1, are useful inthe treatment of edema, hypokalemia, and hyponatremia in edema-formingstates such as liver cirrhosis, congestive heart failure, nephroticsyndrome and renal failure.

[0103] To evaluate whether acute tachyphylaxis develops during i.v.treatment with Compound 1, an additional series of experiments wereperformed. Chronically instrumented rats were infused with vehicle (150mM glucose) or Compound 1 (1 nmol/kg/min) for twelve hours while waterbalance was maintained using a computer-driven, servo-controlled i.v.volume replacement system^([2]). Acute tachyphylaxis has been observedduring repeated i.v. dosing of the vasopressin antagonist SKF 101926 inconscious dogs^([12]) and tolerance during chronic treatment has beenreported for the selective V₂-receptor antagonist OPC-31260 ^([13]).

[0104] Compound 1 elicited a sustained aquaretic response lasting theentire 12-hour infusion period suggesting that acute tachyphylaxis doesnot develop during treatment with this compound. I.v. infusion of 1nmol/kg/min of Compound 1 elicited an aquaretic response of similarmagnitude to the response seen with maximal doses of the selectivevasopressin type-2 receptor antagonist OPC-31260 (FIG. 9) when tested inthe same model^([2]), In this experiment, 1 nmol/kg/min of Compound 1inhibited tubular water reabsorption without concomitant changes inarterial blood pressure, heart rate, renal blood flow, glomerularfiltration rate or proximal tubular reabsorption. These experimentsdemonstrated that Compound 1 has a maximal aquaretic efficacy similar towhat can be obtained with vasopressin type-2 antagonists. However, noacute tachyphylaxis develops within the first 12 hours of treatment withCompound 1 and this finding represents a further therapeutic advantageof Compound 1 relative to vasopressin type-2 receptor antagonists.Moreover, vasopressin type-2 receptor antagonists do not affect renalhandling of electrolytes, while compounds of the present inventiondecrease urinary excretion of sodium and potassium.

[0105] To examine the effect of compounds of the present invention in anin vivo model of heart failure a chronic model of low-output heartfailure will be used. Briefly, the rats will be anaesthetized in aninhalation chamber with 4% halothane in 1:1 N₂O/O₂ gas mixture. Afterinsertion of an endotracheal tube the animal is artificially ventilatedwith 1% halothane in a 1:1 N₂O/O₂ gas mixture. Tidal volume andrespiratory rate are adjusted to maintain arterial pH between 7.35 and7.45. During surgery the animal is placed on a heated table thatmaintained rectal temperature at 37-38° C. Left coronary arteryligation, used to produce congestive heart failure is performed via aparasternal thoracotomy and a 6-0 silk suture is placed between thepulmonary trunk and the left auricle. Sham-operation is performedwithout ligating the left coronary artery. To minimize postsurgicalpain, rats are treated postoperatively with buprenorphine 0.2 mg/kg s.c.b.i.d. for 2 days. Two weeks after heart surgery permanent Tygoncatheters are inserted into the femoral artery and vein, and a bladdercatheter is inserted into the urinary baldder. To prevent coagulation invascular catheters, these lines are filled with 50% dextrose with 1,000units heparin/ml and 10,000 units streptokinase/mi. Inventors of thepresent invention have previously described the use of thismodel^([14]).

[0106] Physiological examinations are performed 3 weeks after heartsurgery since the functional deterioration after left coronary arteryligation is generally maximal at this time. Renal function studies areperformed as described above. To evaluate the degree of heart failure,the rat is anaesthetized with halothane in 1:1 N₂O/O₂, intubated andartificially ventilated, and a Tygon catheter is inserted into the leftventricle via the right carotid artery for measurement of leftventricular end-diastolic pressure (LVEDP). Accurate measurement ofLVEDP is performed by adjusting the concentration of halothanetomaintain after-load during anaesthesia at the same level as meanarterial pressure recorded in the conscious state. This animal modelshares clinical features of low cardiac output, sodium and waterretention, and edema formation with the most common form of human heartfailure^([15]).

[0107] To examine the effect of compounds of the present invention in anin vivo model of nephritic syndrome a chronic model ofadriamycin-induced nephritic syndrome will be used. Nephrotic syndromeis induced with a single intravenous injection of adriamycin(=doxorubicin, Sigma Chemical, St. Louis, Mo.); 7-8 mg/kg at aconcentration of 10 mg/ml dissolved in normal saline. In this model,proteinuria begins 4 to 5 days after a single intravenous injection of7.5 mg/kg. The full expression of the syndrome with glomerular changes,proteinuria, sodium and water retention, and edema-formation develops 13to 15 days later^([15,16]).

[0108] To examine the effect of compounds of the present invention in anin vivo model of liver cirrhosis a chronic model of biliary cirrhosisinduced by common bile duct ligation will be used^([17]). After 28 daysof obstruction, the rats develop a progressive cirrhosis that isassociated with sodium and water retention, and ascites^([15]).

[0109] To examine the effect of compounds of the present invention in anin vivo model of multiple organ failure including acute renal failure, amodel of organ failure elicited by hemorrhage during anesthesia andsurgery will be used. In this model, rats are infused with isotonicsaline or compounds of the present invention for 15 min prior toanesthesia. Then the animals are anestetized with isoflurane (3% inO₂/N₂O mixture) and subjected to periods of surgery (chronic bladdercatheterization+ femoral vein and artery; 30 min); hemorrhage (20 cc/kgb.w.; 45 min), and recovery (blood replacement; 120 min). Consecutive 10min urine samples are collected throughout, and rats are allowed torecover for 7 days. Following the hemorrhagic event, urine collectionsand blood samples are collected (e.g., days 2, 4, and 6) to evaluate therecovery as determined by urine production, and serum concentrations ofcreatinine and urea. Finally on day 7, rats are sacrified forhistological examination of all organs. Using this model, Kapusta et al.previously demonstrated that the kappa opioid agonist U-50,488H preventsmultiple organ failure (kidney, lungs, intestines) and increasessurvival after a hemorrhagic event elicited during surgicalanaesthesia^([18]).

[0110] In summary, in vivo studies have demonstrated that Compound 1 isa potent sodium and potassium-sparing aquaretic. In addition to itsperipheral effects, nociceptin elicits a centrally mediated decrease insympathetic nerve activity, arterial blood pressure and heartrate^([10;19,20]). Moreover, nociceptin decreases spinal sympatheticoutflow by depressing the activity of spinal preganglionic neurons bothin vitro and in vivo^([21]).The rapid onset and similar timerelationship of decreased sympathetic nerve activity, blood pressure andheart rate in rats with sinoaortic baroreceptor denervation suggest thatthe cardiovascular effects of nociceptin is largely mediated byinhibition of sympathetic nerve activity to the cardiovascularsystem^([10]).Thus, the inhibitory action of Compound 1 onnociceptin-induced hypotension and bradycardia suggests that Compound 1is able to antagonize the effects of nociceptin on neurogenic control ofheart rate and blood pressure. The finding that Compound 1 acts as anociceptin antagonist in vivo is supported by the fact that Compound 1antagonizes the smooth muscle relaxing effect of nociceptin in the mousevas deferens model. Thus, in contrast to endogenous nociceptin, Compound1 increases urine flow rate and decreases urinary excretion of sodiumand potassium in doses that do not produced marked bradycardia andhypotension.

[0111] In Vitro Studies of the Binding of Compound 1 to ORL1

[0112] In a series of binding assays we have demonstrated that Compound1 binds to the human nociceptin receptor (i.e, ORL1) with high affinityand high specificity.

[0113] Using HEK293 cells that were transfected with the human ORL1receptor, we have demonstrated that Compound 1 displaces the specificbinding of [3H]-labeled nociceptin with a K_(n)=0.26 nM.

[0114] In addition, Compound 1 has been tested in 5 opioid(non-selective opiate, δ, K, μ,&ORL1), 3 vasopressin (V_(1a), V_(1b) andV₂) receptor binding assays at 0.1, 10, and 1000 nM as well as in 40different receptor, uptake, and ion channel binding sites (A₁,A₂,α₁,α₂,β₁,NE uptake, AT₁, BZD, B₂(h), CCK_(A)(˜CCK₁), D1, D2,ET_(A)(h), GABA, NMDA, H₁, M, NK₁(h), Y, N, opiate, PCP, 5-HT,5-HT_(1B), 5-HT_(2A), 5-HT uptake, σ, V₁) at 10 μM. These experimentsdemonstrated that Compound 1 is a selective ORL1 receptor ligand withnone or only weak binding to other receptors than the ORL1 receptor.Moreover, in transfected HEK293 cells that express the human ORL1receptor, Compound 1 inhibits forskoline-induced formation of cAMP withan EC₅₀=0.54 nM and a relative maximum relative to nociceptin of 92%.These results suggest that Compound 1 is a selective ORL1 receptoragonist.

[0115] Data showing the binding of compounds of the invention to thehuman ORL1 receptor expressed in CHO cells calculated as IC₅₀ (nM)values (all based on one experiment) are shown in Table 6 below. TheORL1 receptor is negatively coupled to cAMP and thus the effect of ORL1agonists are detected as an inhibition of forskolin-induced cAMPformation. Six compounds of the invention have been tested for efficacyon hORL1 receptor expressed in CHO cells. The EC₅₀ (nM) values are allbased on one experiment (data shown in Table 6). TABLE 6 Binding ofZS-compounds (peptides) on hORL1 receptor Compound number Binding IC₅₀(nM) Efficacy EC₅₀ (nM) CE1 0.31 0.41 1 0.32 0.58 3 235 83 4 ND 91 6 115135 7 >100 ND 8 78 59 9 3.7 0.035 10 0.51 ND 11 0.27 ND 12 0.15 ND 13 NDND

[0116] Most of the published work concerning the human ORL1 receptor hasbeen focused on the CNS effects of the receptor. However, the workdescribed herein is focused on the effect mediated by Compound 1 in thekidney. In order to determine whether the ORL1 receptor is present inthe human kidney, we have constructed PCR primers homologous to the ORL1receptor expressed in the human brain. Total RNA isolated from humankidney RNA was subjected to first strand synthesis to obtain materialfor PCR analysis. This analysis showed a 449 bp fragment of the ORL1receptor indicating that the receptor is expressed in the human kidney,and therefore, ORL1 ligands of the present are expected to have renalactions in humans.

[0117] In Vitro Stability

[0118] Degradation of peptides is almost exclusively seen in thepresence of enzymes, e.g. in the bloodstream. An important feature of aputative drug is whether it is quickly degraded in vivo or whether it isable to conduct its effects over a longer period of time. To be able toexamine the stability of the peptide conjugates of the present inventiondegradation was tested in heparin stabilised mouse plasma (obtained fromHarlan Seralab, UK).

[0119] Methods

[0120] The stability studies were run in triplicate. 100 μL of a 1 mg/mLsolution of the test compound (peptide conjugate) was added to anEppendorf tube, containing 900 μL plasma, incubated at 37° C.Immediately after addition, a sample of 100 μL was removed and added toan Eppendorf tube, containing 10 μL of extraction solution (50% TFA(trifluoroacetic acid) in MeCN, v/v). This sample constitutes t=0.Subsequently, samples were removed at t=15, 30, 60, 120, 180, 240, 300,and 360 min and treated in the same manner as for t=0. The samples arecentrifuged at 20,000×g, and the supernatant was transferred to vialsand analysed by high-performance liquid chromatography (HPLC).

[0121] The analysis was performed on a HP1100 HPLC with a quaternarypump. The column used was a Vydac 201SP5215, and the apparatus comprisesan autosampler, a column oven, a variable UV detector and a fluorescencedetector. The process was controlled by ChemStation software. Thegradient elution consisted of two solvents A and B, which contained0.02% n-Heptafluorobutyric acid (HFBA) in MilliQ water (MQW) and 0.02%HFBA in 90% MeCN in MQW, respectively. The separation was performed at30° C. using a linear gradient from 5-100% B in 15 minutes at a flow of0.250 mL/min. The chromatograms used in this study were obtained by UVdetection at a wavelength of 254 nm. The peaks identified as the testpeptides by comparison to known standards were integrated to yield peakarea as a measure of concentration for each time point. These valueswere transferred to Microsoft Excel, where half-lives (t½) and rateconstant (k_(obs)) for Ac-RYYRWK-NH₂ and Compound 1 were determined byfitting to the equation. As illustrated in FIG. 10 the rate ofdegradation of the unconjugated substance Ac-RYYRWK-NH₂ wassignificantly faster than for Compound 1 (Ac-RYYRWK-NH₂; t½=2−5 min(estimated) and Compound 1:t½=358 +/−36 min). These data demonstratethat the conjugated peptide Compound 1 is significantly more stable inheparinized mouse plasma than the unconjugated hexapeptide,Ac-RYYRWK-NH₂. Thus, the invention also relates to a method of enhancingthe stability of a hexapeptide, preferably a hexapeptide of formula IIherein, and more preferably the hexapeptide RYYRWK, by linking a peptidesequence Z′ as defined herein, preferably Z′ is a K₆ sequence, via apeptide bond to the C-terminal of said hexapeptide.

[0122] Stability in Heparinised Human Plasma

[0123] In addition, the degradation of Compound 1 of the invention hasbeen studied in heparinised human plasma. Table 7 below shows the halflife of degradation (T½) of Compound 1 of the invention compared toAc-RYYRWK-NH2 and nociceptin-NH2. It appears from the table thatCompound 1 of the invention having a half life of more than 16 hours isconsiderably more stable in plasma than Ac-RYYRWK-NH2 which has a halflife of less than 1 hour, and nociceptin-NH₂ which has a half life ofabout 1.5 hours. TABLE 7 Results of in vitro stability test in plasmaand serum, T_(1/2) in minutes (mean ± deviation). T_(1/2) in minCOMPOUNDS (degradation in human heparinised plasma) Compound 1  973.8min ± 21.9% Ac-RYYRWK-NH₂    50.7 min ± 20.9% Nociceptin-NH₂  97.5 min ±6.4%

[0124] Method of Analysis of in Vitro Plasma Stability

[0125] The stability of peptides is analysed in human plasma. Thepeptides are incubated at 37° C. in plasma and samples taken at approx.8 regular intervals between t=0 and t=390 min for Compound 1 andnociceptin-NH₂, and at 9 intervals between t=0 and t=32 min for CompoundCE1 are analysed by HPLC. Appropriate conditions (column, solvent,gradient, and temp.) for the HPLC analyses are estimated to ensure thatthe drug peak and the plasma peaks do not have the same retention time.This is done by subsequent injections of the drug, plasma, and aco-injection with the drug and the plasma, followed by optimisation ofthe LC method parameters until a satisfactory separation is obtained.Three parallel experiments are performed for each plasma type. 100 μl ofpeptide is mixed with 900 μl plasma at t=0 and incubated at 37° C.(drug-plasma mixture conc. 0.1 mg/ml). Samples of 100 μl of thedrug-plasma mixture are removed at appropriate intervals and thedegradation stopped by precipitation of the sample with 10 μl MeCN:TFA50:50 v/v. A control plasma sample without the drug treated in the samemanner is also taken. The plasma samples are centrifuged for 15 min. at12,000 rpm (Eppendorf centrifuge) at ambient temperature. The resultingsupernatant solution is transferred to 300 μl HP autosampler vials andanalyzed by HPLC. HPLC analysis are performed as follows:

[0126] Ac-RYYRWK-NH₂ and Compound 1

[0127] Column: Vydac 201 SP5215, 991203, 033,

[0128] S/N N 905550-1-3, 150×2.1 mm, flow: 0.200 mL/min. Temp.: 40° C.

[0129] Solvent A: 0.02% HFBA in MQW. Solvent B: 0.02% HFBA in MQW:MeCN10:90

[0130] Run time=25 min. Inj.vol.:20 μL. Detection: FLD1 A, Ex.=280 nm,Em.=335 nm

[0131] Gradient (time in min; %B): 0;5 5;50 15;100 15.5;5 25;5

[0132] Nociceptin-NH₂

[0133] Column: Vydac 218MSP52, 000517, 095,

[0134] S/N N 970520-9-7, 250×2.1 mm, flow: 0.200 mL/min. Temp.: 40° C.

[0135] Solvent A: 0.1% TFA in MQW. Solvent B: 0.1% TFA in MQW:MeCN 10:90

[0136] Run time=25 min. Inj.vol.:20 μL. Detection: MWD Signal=215.16 nm,ref.=360, 100nm

[0137] Gradient (time in min; %B): 0;1 5;1 15;40 16;100 17;1 25;1

[0138] The samples are analysed in the following order: blank, thepeptide at 0.1 mg/mL, the plasma without the peptide, the three parallelsamples for t=0, the three parallel samples for t=5 min. the threeparallel samples for t=60 min. etc. And finally the three parallelsamples for t=0 are repeated to make sure that there have been nodegradation or other failure during the analyses. The sampleconcentrations (peak height in mAU) are plotted vs. time and fitted to afunction describing a mono exponential decay (Excel). The half-lives ofthe peptides in plasma are presented in Table 7 as mean (n=3)± standarddeviation.

[0139] Compositions

[0140] The invention also concerns a composition comprising apharmacologically active peptide conjugate as defined herein incombination with a pharmaceutically acceptable carrier and/or diluent.Such compositions may be in a form adapted to oral, subcutaneous,parenteral (intravenous, intraperitoneal), intramuscular, rectal,epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocularly,or pulmonary administration, preferably in a form adapted foradministration by a peripheral route, or is suitable for oraladministration or suitable for parenteral administration. Otherpreferred routes of administration are subcutaneous, intraperitoneal andintravenous, and such compositions may be prepared in a mannerwell-known to the person skilled in the art, e.g., as generallydescribed in “Remington's Pharmaceutical Sciences”, 17. Ed. Alfonso R.Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 andmore recent editions and in the monographs in the “Drugs and thePharmaceutical Sciences” series, Marcel Dekker. The compositions mayappear in conventional forms, for example, solutions and suspensions forinjection, capsules and tablets, preferably in the form of entericformulations, e.g. as disclosed in U.S. Pat. No. 5,350,741, for oraladministration.

[0141] The pharmaceutical carrier or diluent employed may be aconventional solid or liquid carrier. Examples of solid carriers arelactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin,acacia, magnesium stearate, stearic acid or lower alkyl ethers ofcellulose. Examples of liquid carriers are syrup, peanut oil, olive oil,phospholipids, fatty acids, fatty acid amines, polyoxyethylene andwater. Similarly, the carrier or diluent may include any sustainedrelease material known in the art, such as glyceryl monostearate orglyceryl distearate, alone or mixed with a wax.

[0142] When a solid carrier is used for oral administration, thepreparation may be tabletted, placed in a hard gelatin capsule in powderor pellet form or it can be in the form of a troche or lozenge. Theamount of solid carrier will vary widely but will usually be from aboutabout 25 mg to about 1 g.

[0143] A Typical Tablet Which may be Prepared by Conventional TablettingTechniques may Contain

[0144] Core: active compound (as free compound or salt thereof) 100 mg;colloidal silicon dioxide (Aerosil) 1.5 mg; cellulose, microcryst.(Avicel) 70 mg; modified cellulose gum (Ac-Di-Sol) 7.5 mg; magnesiumstearate. Coating: HPMC approx. 9 mg; Mywacett 9-40T (acylatedmonoglyceride used as plasticizer for film coating) approx. 0.9 mg.

[0145] When a liquid carrier is used, the preparation may be in the formof a syrup, emulsion, soft gelatin capsule or sterile injectable liquidsuch as an aqueous or non-aqueous liquid suspension or solution.

[0146] The composition may also be in a form suited for local orsystemic injection or infusion and may, as such, be formulated withsterile water or an isotonic saline or glucose solution. It is preferredthat the compositions of the invention are in a form adapted forperipheral administration only, with the exeption of centrallyadministrable forms.

[0147] The compositions may be sterilized by conventional sterilizationtechniques which are well known in the art. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with thesterile aqueous solution prior to administration. The composition maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents and the like, for instance sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0148] Formulation of Peptide Conjugate for Intravenous Injection

[0149] Multi-dose formulations may be prepared as a solution of acompound of the invention in sterile, isotonic saline, stored in cappedvials, and if necessary a preservative is added (e.g. benzoates). Fixeddose formulations may be prepared as a solution of the compound insterile, isotonic saline, stored in glass ampoules, and if necessaryfilled with an inert gas. Each dose of the compound is stored dry inampoules or capped vials, if necessary filled with inert gas. Themulti-dose formulation demands the highest degree of stability of thecompound. When the stability of the compound is low fixed doseformulations can be used.

[0150] For nasal administration, the preparation may contain a compoundof the present invention dissolved or suspended in a liquid carrier, inparticular, an aqueous carrier, for aerosol application. The carrier maycontain additives such as solubilizing agents, e.g, propylene glycol,surfactants such as bile acid salts or polyoxyethylene higher alcoholethers, absorption enhancers such as lecithin (phosphatidylcholine) orcyclodextrin, or preservatives such as parabines.

[0151] In a preferred embodiment of the invention the compound offormula I, II or III is administered as a dose in the range from about0.001 to about 10 g per patient per day, preferably from about 1 toabout 1000 mg per patient per day, more preferably from about 10 toabout 100 mg per patient per day, about 50 mg per patient per day. Apharmaceutical composition adapted for oral administration typicallycontains in a unit dosage an amount of a compound of formula I or IIranging from about 1 to about 100 mg. A pharmaceutical compositionadapted for parenteral administration typically contains in a unitdosage an amount of a compound of formula I or II ranging from about 0.1to about 10 mg.

[0152] The invention also concerns a pharmacologically active compoundwhich is a peptide conjugate or a derivative or salt thereof asdisclosed herein for use in therapy, and the use thereof as definedherein for the manufacture of a pharmaceutical composition for use intherapy, e.g., in the treatment of ORL1 related peripheral diseases andailments. Preferably, a pharmaceutical composition is suitale for oraladministration. Therapeutic uses of the novel peptide conjugates hereinare to increase the renal excretion of water and to decrease urinarysodium and potassium excretion (i.e., a selective water diuresis) usingrelatively low doses of the active compound, i.e. preferably less than50 mg per patient per day, and to decrease blood pressure in absence ofreflex tachycardia and increase appetite using higher doses of theactive compound.

[0153] In specific embodiments, a peptide conjugate according to thepresent invention may be used to avoid CNS side effects due to poorpenetration of the peptide conjugates into the central nervous system,in the treatment of specific diseases or ailments amenable to treatmentwith compounds having nociceptin-like activity.

[0154] In specific embodiments, a peptide conjugate according to thepresent invention may be used as an agonist or partial agonist or as aninhibitor depending on clinical utility acting on a nociceptin receptor,such as ORL1, especially where said receptor is found in peripheraltissue. Thus, the peptide conjugate of formula I or III is useful forthe preparation of a medicament to be used in the treatment of diseasestates associated with elevated tone of nociceptin.

[0155] In specific embodiments, a peptide conjugate according to thepresent invention may be used for the preparation of a medicament forthe treatment and/or prevention of hyponatremia and/or hypokalemia aswell as in a method of treating and/or preventing hyponatremia and/orhypokalemia.

[0156] Furthermore, a peptide conjugate according to the presentinvention may be used for the preparation of a medicament for thetreatment and/or prevention of sodium and water retaining conditions andacute renal failure as well as in a method of treating and/or preventingmultiple organ failure and sodium and water retaining conditions andacute renal failure. Furthermore, a peptide conjugate according to thepresent invention may be used for the preparation of a medicament forthe treatment and/or prevention of organ failure conditions associatedwith extracellular fluid volume expansion which include:

[0157] 1. Congestive heart failure in which the heart failure may bedescribed as systolic or diastolic, high-output or low-output, acute orchronic, right-sided or left-sided, and forward or backward. An exampleof a predictive in vivo heart failure model for the study of therapeuticactions of peptides of the present invention is the conscious rat modelof low-cardiac output induced by ligation of the left coronaryartery^([15]) which is incorporated by reference.

[0158] 2. Liver cirrhosis in which the cirrhosis may be related toalcoholic liver disease; postnecrotic cirrhosis (caused by infectiousdiseases, inherited metabolic disorders, drugs and toxins, inflammatoryand other diseases); biliary cirrhosis (primary or secondary); cardiaccirrhosis due to prolonged, severe right-sided congestive heart failure;metabolic, hereditary, drug-related or other types of cirrhosis. Anexample of a predictive in vivo model of liver cirrhosis for the studyof therapeutic actions of peptides of the present invention is theconscious rat model of liver cirrhosis induced by ligation of the commonbile duct^([15]).

[0159] 3. Nephrotic syndrome related to systemic and/or renal disease,drug- or toxin-induced. An example of a predictive in vivo model ofnephritic syndrome for the study of therapeutic actions of peptides ofthe present invention is the conscious rat model of nephritic syndromeinduced by i.v. administration of adriamycin^([15]).

[0160] 4. Hypertension in which the hypertension may be primary(idiopathic) or secondary to drugs, toxins or diseases in endocrineglands, kidneys, or in the central nervous system. Examples ofpredictive in vivo models of hypertension for the study of therapeuticactions of peptides of the present invention is the spontaneouslyhypertensive rat or the Dahl salt-sensitive rat.

[0161] 5. Multiple organ failure elicited during hemorrhagic shockincluding acute renal failure. An example of a predictive in vivo modelof multiple organ failure (kidney, lungs, intestines) elicited duringanaesthesia, surgery and hemorrhage is described by Kapusta etal.^([18]) which is incorporated by reference.

[0162] 6. Acute renal failure in which the pathogenesis of the diseasemay be related to either prerenal or intrinsic renal causes:

[0163] Prerenal azotemia, such as the following conditions:

[0164] I. Hypovolemia

[0165] A. Hemorrhage, burns, dehydration

[0166] B. Gastrointestinal fluid loss: vomiting, surgical drainage,diarrhea

[0167] C. Renal fluid loss: diuretics, osmotic diuresis (e.g., diabetesmellitus), hypoadrenalism

[0168] D. Sequestration in extravascular space: pancreatitis,peritonitis, trauma, burns, severe hypoalbuminemia;

[0169] II. Low cardiac output

[0170] A. Diseases of myocardium, valves, and pericardium, arrhythmias,tamponade

[0171] B. Other: pulmonary hypertension, massive pulmonary embolus,positive pressure mechanical ventilation;

[0172] III. Altered renal systemic vascular resistance ratio

[0173] A. Systemic vasodilatation: sepsis, antihypertensives, afterloadreducers, anesthesia, anaphylaxis

[0174] B. Renal vasoconstriction: hypercalcemia, norepinephrine,epinephrine, cyclosporine, amphotericin B

[0175] C. Cirrhosis with ascites (hepatorenal syndrome);

[0176] IV. Renal hypoperfusion with impairment of renal autoregulatoryresponses

[0177] Cyclooxygenase inhibitors, angiotensin-converting enzymeinhibitors;

[0178] V. Hyperviscosity syndrome (rare)

[0179] Multiple myeloma, macroglobulinemia, polycythemia.

[0180] Acute renal failure further include:

[0181] Intrinsic renal azotemia, such as the following conditions:

[0182] I. Renovascular obstruction (bilateral or unilateral with onefunctioning kidney)

[0183] A. Renal artery obstruction: atherosclerotic plaque, thrombosis,embolism, dissecting aneurysm, vasculitis

[0184] B. Renal vein obstruction: thrombosis, compression;

[0185] II. Disease of glomeruli or renal microvasculature

[0186] A. Glomerulonephritis and vasculitis

[0187] B. Hemolytic uremic syndrome, thrombotic thrombocytopenicpurpura, disseminated intravascular coagulation, toxemia of pregnancy,accelerated hypertension, radiation nephritis, systemic lupuserythematosus, scleroderma;

[0188] III. Acute tubular necrosis

[0189] A. Ischemia: as for prerenal azotemia (hypovolemia, low cardiacoutput, renal vasoconstriction, systemic vasodilatation), obstetriccomplications (abruptio placentae, postpartum hemorrhage)

[0190] B. Toxins

[0191] 1. Exogenous: radiocontrast, cyclosporine, antibiotics (e.g.,aminoglycosides), chemotherapy (e.g., cisplatin), organic solvents(e.g., ethylene glycol), acetaminophen, illegal abortifacients

[0192] 2. Endogenous: rhabdomyolysis, hemolysis, uric acid, oxalate,plasma cell dyscrasia (e.g., myeloma);

[0193] IV. Interstitial nephritis

[0194] A. Allergic: antibiotics (e.g., -lactams, sulfonamides,trimethoprim, rifampicin), nonsteroidal anti-inflammatory agents,diuretics, captopril

[0195] B. Infection: bacterial (e.g., acute pyelonephritis,leptospirosis), viral (e.g., cytomegalovirus), fungal (e.g.,candidiasis)

[0196] C. Infiltration: lymphoma, leukemia, sarcoidosis

[0197] D. Idiopathic;

[0198] V. Intratubular deposition and obstruction

[0199] A. Myeloma proteins, uric acid, oxalate, acyclovir, methotrexate,sulphonamides;

[0200] VI. Renal allograft rejection

[0201] Examples of predictive in vivo models of prerenal azotemia forthe study of therapeutic actions of peptides of the present inventionare the norepinephrine and renal artery clamp rat ischemic acute renalfailure models, that may be accelerated by hemorrhage^([22]) which isincorporated by reference.

[0202] An example of a predictive in vivo model of intrinsic renalazotemia for the study of therapeutic actions of peptides of the presentinvention is the gentamicin-induced acute renal failure model^([23])which is incorporated by reference.

REFERENCES

[0203] [1.] C. T. Dooley, C. G. Spaeth, I. P. Berzetei-Gurske, K.Craymer, I. D. Adapa, S. R. Brandt, R. A. Houghten, L. Toll, JPharmacol.Exp.Ther. 1997, 283 735-741.

[0204] [2.] T. E. Jonassen, S. Christensen, T. H. Kwon, S. Langhoff, N.Salling, S. Nielsen, Am J Physiol Renal Physiol 2000, 279 F1101-F1109.

[0205] [3.] E. Braunwald, in Harrison's Principles of INternalMedicineEds.: A. S. Fauci, E. Braunwald, K. J. Isselbacher, J. D.Wilson, J. B. Martin, D. L. Kasper, S. L. Hauser, D. L. Longo),McGraw-Hill, New York 1998, p. pp. 1287-1298.

[0206] [4.] J. Hughes, H. W. Kosterlitz, F. M. Leslie, Br.J Pharmacol.1975, 53 371-381.

[0207] [5.] Smith, C. B. NIDA Research Monograph. [76], 288-294. 1987.Ref Type: Serial (Book,Monograph)

[0208] [6.] G. Calo, A. Rizzi, G. Bogoni, V. Neugebauer, S. Salvadori,R. Guerrini, C. Bianchi, D. Regoli, Eur.J PharmacoL 1996, 311 R3-R5.

[0209] [7.] Kenakin, T. Pharmacological Analysis of Drug-ReceptorInteraction. 2nd. 1993. USA, Raven Press. Ref Type: Serial(Book,Monograph)

[0210] [8.] D. R. Kapusta, S. F. Sezen, J. K. Chang, H. Lippton, V. A.Kenigs, Life Sci. 1997, 60 L15-L21.

[0211] [9.] D. R. Kapusta, Peptides 2000, 21 1081-1099.

[0212] [10.] D. R. Kapusta, V. A. Kenigs, Am J Physiol 1999, 277R987-R995.

[0213] [11.] H. C. Champion, R. L. Pierce, P. J. Kadowitz, Regul.Pept.1998, 7869-74.

[0214] [12.] G. G. Liversidge, C. G. Wilson, W. L. Sternson, L. B.Kinter, J Appl.Physiol 1988, 64 377-383.

[0215] [13.] T. E. N. Jonassen, S. Christensen, J. S. Petersen,J.Am.Soc.Nephrol. 1997, 8 19A.

[0216] [14.] S. Nielsen, J. Terris, D. Andersen, C. Ecelbarger, J.Frokiaer, T. Jonassen, D. Marples, M. A. Knepper, J. S. Petersen,Proc.Natl.Acad.Sci.U.S.A 1997, 94 5450-5455.

[0217] [15.] G. F. DiBona, P. J. Herman, L. L. Sawin, Am J Physiol 1988,254 R1017-R1024.

[0218] [16.] T. Bertani, A. Poggi, R. Pozzoni, F. Delaini, G. Sacchi, Y.Thoua, G. Mecca, G. Romuzzi, M. B. Donati, Lab Invest 1982, 46 16-23.

[0219] [17.] J. Kountouras, B. H. Billing, P. J. Scheuer, Br.JExp.Pathol. 1984, 65 305-311.

[0220] [18.] D. R. Kapusta, V. A. Kenigs, L. A. Dayan, A. W. Dreisbach,S. Meleg-Smith, V. Batuman, K. J. Varner, J.Am.Soc.Nephrol. 2000, 11A3175.

[0221] [19.] T. Shirasaka, T. Kunitake, K. Kato, M. Takasaki, H. Kannan,Am J Physiol 1999, 277 R1025 R1032.

[0222] [20.] D. R. Kapusta, J. K. Chang, V. A. Kenigs, JPharmacol.Exp.Ther. 1999, 289 173-180.

[0223] [21.] C. C. Lai, S. Y. Wu, C. T. Chen, N. J. Dun, Am J PhysiolRegul.Integr Comp. Physiol 2000, 278 R592-R597.

[0224] [22.] J. D. Conger, M. F. Schultz, F. Miller, J. B. Robinette,Kidney Int. 1994, 46 318-323.

[0225] [23.] D. de Rougemont, A. Oeschger, L. Konrad, G. Thiel, J.Torhorst, M. Wenk, P. Wunderlich, F. P. Brunner, Nephron 1981, 29176-184.

[0226] Preparation of Peptides

[0227] The peptides or peptide conjugates herein are preferably preparedusing peptide synthetic methods but may also be prepared by means ofrecombinant DNA-technology using general methods and principles known tothe person skilled in the art. Thus the present invention also relatesto a nucleic acid sequence encoding a polypeptide sequence comprisingthe peptide sequence of formula I or Ill; a vector carrying said nucleicacid sequence, and a host cell comprising said nucleic acid sequence andcapable of expressing said polypeptide sequence.

[0228] A nucleic acid sequence encoding the present peptide conjugatemay be prepared synthetically by established standard methods, e.g., thephosphoamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., EMBO Journal 3, 1984, pp. 801-805. According to thephosphoamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned insuitable vectors. The techniques used to isolate or clone a nucleic acidsequence encoding the peptide X are known in the art and includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof. The cloning of the nucleic acid sequences of the presentinvention from such genomic DNA can be effected, e.g., by using the wellknown polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, A Guide to Methodsand Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligatedactivated transcription (LAT) and nucleic acid sequence-basedamplification (NASBA) may be used. It can then be ligated to a nucleicacid sequence encoding Z.

[0229] The nucleic acid sequence encoding the conjugate is then insertedinto a recombinant expression vector which may be any vector which mayconveniently be subjected to recombinant DNA procedures. The choice ofvector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated. Inthe vector, the nucleic acid sequence encoding the conjugate of thepresent invention should be operably connected to a suitable promotersequence. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice and may be derivedfrom genes encoding proteins either homologous or heterologous to thehost cell. Examples of suitable promoters for directing thetranscription of the nucleic acid sequence encoding the conjugate,especially in a bacterial host cell, are the promoters obtained from theE. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), andthe prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75:3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80:21 25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242:74-94; and in Sambrook et al., 1989, supra. In ayeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423488.

[0230] The nucleic acid sequence encoding said conjugate may also beoperably connected to a suitable terminator, such as the human growthhormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814).Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase.

[0231] The vector may further comprise elements such as polyadenylationsignals (e.g., from SV 40 or the adenovirus 5 Elb region),transcriptional enhancer sequences (e.g., the SV 40 enhancer) andtranslational enhancer sequences (e.g., the ones encoding adenovirus VARNAs). Useful polyadenylation sequences for yeast host cells aredescribed by Guo and Sherman, 1995, Molecular Cellular Biology15:5983-5990. The recombinant expression vector may further comprise aDNA sequence enabling the vector to replicate in the host cell inquestion. Examples of such a sequence (when the host cell is a mammaliancell) is the SV 40 or polyoma origin of replication. Examples ofbacterial origins of replication are the origins of replication ofplasmids pBR322, pUC19, pACYC177, pACYC184, pUB 10, pE194, pTA 060, andpAMβ1. Examples of origin of replications for use in a yeast host cellare the 2 micron origin of replication, the combination of CEN6 andARS4, and the combination of CEN3 and ARS1. The origin of replicationmay be one having a mutation to make its function temperature-sensitivein the host cell (see, e.g., Ehrlich, 1978, Proc. Natl. Acad. Sci. USA75:1433).

[0232] The vector may also comprise a selectable marker, e.g., a genethe product of which complements a defect in the host cell, such as thegene coding for dihydrofolate reductase (DHFR) or one which confersresistance to a drug, e.g., neomycin, geneticin, ampicillin, orhygromycin. Suitable markers for yeast host cells are ADE2, HIS3, LEU2,LYS2, MET3, TRP1, and URA3.

[0233] The procedures used to ligate the nucleic acid sequences codingfor the conjugate, the promoter and the terminator, respectively, and toinsert them into suitable vectors containing the information necessaryfor replication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.). The host cell into which theexpression vector is introduced may be any cell which is capable ofproducing the conjugate and it may be a eukaryotic cell, such asinvertebrate cells or vertebrate cells, e.g., Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the COS, BHK or CHO cell lines.Methods for transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are described in e.g., Kaufman and Sharp, 1982,J. Mol. Biol. 159:601-621; Southern and Berg, 1982, J. Mol. Appl. Genet.1:327-341; Loyter et al., 1982, Proc. Nat Acad. Sci. USA 79:422-426;Wigler et al., 1978, Cell 14:725; Corsaro and Pearson, 1981, SomaticCell Genetics 7:603, Graham and van der Eb, 1973, Virology 52:456;Fraley et al., 1980, JBC 225:10431; Capecchi, 1980, Cell 22:479; Wiberget al., 1983,NAR 11:7287; and Neumann et al., 1982, EMBO J. 1:841-845.

[0234] The host cell may also be a unicellular pathogen, e.g., aprokaryote, or a non-unicellular pathogen, e.g., a eukaryote. Usefulunicellular cells are bacterial cells such as gram positive bacteriaincluding, but not limited to, a Bacillus cell or a Streptomyces cell,or gram negative bacteria such as E. coli and Pseudomonas sp. Thetransformation of a bacterial host cell may, for instance, be effectedby protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168:111-115), by using competent cells (see,e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, orDubnar and Davidoff Abelson, 1971, Journal of Molecular Biology56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169:5771-5278).

[0235] The host cell may be a fungal cell, e.g., Neurospora,Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium(=Aspergillus). The fungal host cell may also be a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The medium used to culture the cells may be anyconventional medium suitable for growing mammalian cells, such as aserum-containing or serum-free medium containing appropriatesupplements, or a suitable medium for growing insect, yeast or fungalcells. Suitable media are available from commercial suppliers or may beprepared according to published recipes (e.g,. in catalogues of theAmerican Type Culture Collection).

[0236] Thus, the invention also relates to a method for producing thepeptide conjugate of formula I or III having a natural polypeptidesequence, comprising

[0237] introducing a nucleic acid sequence encoding a polypeptidesequence comprising the peptide sequence of formula I or III and aselectable marker contained within a nucleic acid construct or a vectorinto a host cell to obtain a recombinant host cell;

[0238] selecting said recombinant host cell;

[0239] culturing said recombinant host cells under conditions permittingthe production of said polypeptide sequence;

[0240] isolating said polypeptide sequence from the culture; and

[0241] optionally cleaving said polypeptide sequence using anappropriate protease to obtain said peptide conjugate.

[0242] The peptide conjugate of formula I or III having a naturalpolypeptide sequence thus prepared may then be recovered from theculture medium by conventional procedures including separating the hostcells from the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g., ammonium sulphate, purification by a variety ofchromatographic procedures, e.g., ion exchange chromatography, affinitychromatography, or the like. Chemical modification steps to obtainnon-natural derivatives may further be employed.

[0243] The peptides of the invention may be prepared by methods knownper se in the art. Thus, the peptide sequences X and Z may be preparedby standard peptide-preparation techniques such as solution synthesis orMerrifield-type solid phase synthesis. Both the Boc(tert.butyloxycarbonyl) as well as the Fmoc(9-fluorenylmethyloxycarbonyl)strategies are applicable.

[0244] Preferred synthetic methods include a method for the preparationof a peptide conjugate of formula IV (X-Z′), comprising the steps of:

[0245] a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the activated form to animmobilised peptide sequence H-Z′-SSM, thereby forming an immobilisedN-α-protected peptide fragment,

[0246] b) removing said N-α-protecting group, thereby forming animmobilised protected peptide fragment having an unprotected N-terminal,

[0247] c) coupling an additional amino acid or dipeptide having suitableprotecting groups including an N-α-protecting group in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and c)until the desired peptide sequence X is obtained, and then

[0248] d) cleaving off the peptide conjugate from the solid supportmaterial;

[0249] and a method for the preparation of a peptide conjugate offormula V (Z-X), comprising the steps of:

[0250] a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the activated form to asolid support material (SSM), thereby forming an immobilised protectedamino acid or a protected dipeptide,

[0251] b) removing said N-α-protecting group, thereby forming animmobilised amino acid or peptide fragment having an unprotectedN-terminal,

[0252] c) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised amino acid orpeptide fragment, and repeating the removal/coupling step procedure instep b) and c) until the desired peptide sequence X is obtained,

[0253] d) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and d)until the desired peptide sequence Z is obtained, and then

[0254] e) cleaving off the peptide conjugate from the solid supportmaterial.

[0255] and, furthermore, a method for the preparation of a peptideconjugate of formula VI (Z-X-Z′), comprising the steps of:

[0256] a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the carboxyl activatedform to an immobilised peptide sequence H-Z′-SSM, thereby forming animmobilised N-α-protected peptide fragment,

[0257] b) removing said N-α-protecting group, thereby forming animmobilised peptide fragment having an unprotected N-terminal,

[0258] c) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and c)until the desired peptide sequence X is obtained, and then

[0259] d) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and d)until the desired peptide sequence Z is obtained, and then

[0260] e) cleaving off the peptide conjugate from the solid supportmaterial.

[0261] Experimental Procedures

[0262] Apparatus and Synthetic Strategy

[0263] Peptides were synthesized batchwise in a polyethylene vesselequipped with a polypropylene filter for filtration using9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting group andsuitable common protection groups for side-chain functionalities.

[0264] Solvents

[0265] Solvent DMF (N,N-dimethylformamide, Riedel de-Häen, Germany) waspurified by passing through a column packed with a strong cationexchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen,Germany) and analyzed for free amines prior to use by addition of3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise toa yellow color (Dhbt-O-anion) if free amines are present. Solvent DCM(dichloromethane, analytical grade, Riedel de-Häen, Germany) was useddirectly without purification. Acetonitril (HPLC-grade, Lab-Scan, DublinIreland) was used directly without purification.

[0266] Amino Acids

[0267] Fmoc-protected amino acids were purchased from Advanced ChemTech(ACT) in suitabel side-chain protected forms.

[0268] Coupling Reagents

[0269] Coupling reagent diisopropylcarbodiimide (DIC) was purchased fromRiedel de-Häen, Germany.

[0270] Solid Supports

[0271] Peptides were synthesized on TentaGel S resins 0.22-0.31 mmol/g.TentaGel S-Ram, TentaGel S RAM-Lys(Boc)Fmoc (Rapp polymere, Germany)were used in cases where a C-terminal amidated peptide was preferred,while TentaGel S PHB, TentaGel S PHB Lys(Boc)Fmoc were used when aC-terminal free carboxylic acid was preferred.

[0272] Catalysts and other Reagents

[0273] Diisopropylethylamine (DIEΛ) was purchased from Aldrich, Germany,piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany.Ethandithiol was purchased from Riedel-de Häen, Frankfurt, Germany.3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH),1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka,Switzerland. Acetic anhydride was obtained from Fluka.

[0274] Coupling Procedures

[0275] The amino acids were coupled as in situ generated HObt or HOAtesters made from appropriate N-α-protected amino acids and HObt or HOAtby means of DIC in DMF. Acylations were checked by the ninhydrin testperformed at 80° C. in order to prevent Fmoc deprotection during thetest (Larsen, B. D. and Holm, A., Int. J. Peptide Protein Res. 43, 1994,1-9).

[0276] Deprotection of the N-α-amino Protecting Group (Fmoc)

[0277] Deprotection of the Fmoc group was performed by treatment with20% piperidine in DMF (1×5 and 1×10 min.), followed by wash with DMF(5×15 ml, 5 min. each) until no yellow color could be detected afteraddition of Dhbt-OH to the drained DMF.

[0278] Coupling of HOBt-esters

[0279] 3 eq. N-α-amino protected amino acid was dissolved in DMFtogether with 3 eq. HObt and 3 eq DIC and then added to the resin.

[0280] Acetylation of the N-terminal Amino Group with Acetic Anhydride

[0281] 40 eq acetic anhydride was dissolved in DMF together with 5 eqpyridine and then added to the resin. The acylation was checked by theninhydrin test as described above.

[0282] Trifluoroacetylation of the N-terminal Amino Group with EthylTrifluoroacetate

[0283] 30 eq ethyl trifluoroacetate was dissolved in dichloromethanetogether with 10 eq triethyl amine and then added to resin. Theacylation was checked by ninhydrin test as described above.

[0284] Cleavage of Peptide from Resin with Acid

[0285] Peptides were cleaved from the resins by treatment with 95%triflouroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany)-water v/vor with 95% TFA and 5% ethandithiol v/v at r.t. for 2 h. The filteredresins were washed with 95% TFA-water and filtrates and washingsevaporated under reduced pressure. The residue was washed with ether andfreeze dried from acetic acid-water. The crude freeze dried product wasanalyzed by high-performance liquid chromatography (HPLC) and identifiedby mass spectrometry (MS).

[0286] Batchwise Peptide Synthesis on TentaGel Resin (PEG-PS)

[0287] TentaGel resin (1 g, 0.23-0.24 mmol/g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtration.The resin was swelled in DMF (15 ml), and treated with 20% piperidine inDMF in order to remove the initial Fmoc group either on the linkerTentaGel S RAM or on the first amino acid on the resin TentaGel SRAM-Lys(Boc)Fmoc. The resin was drained and washed with DMF until noyellow color could be detected after addition of Dhbt-OH to the drainedDMF. The amino acids according to the sequence were coupled as preformedFmoc-protected HObt esters (3 eq.) as described above. The couplingswere continued for 2 h, unless otherwise specified. The resin wasdrained and washed with DMF (5×15 ml, 5 min each) in order to removeexcess reagent. All acylations were checked by the ninhydrin test asdescribed above. After completed synthesis the peptide-resin was washedwith DMF (3×15 ml, 5 min each), DCM (3×15 ml, 1 min each) and finallydiethyl ether (3×15 ml, 1 min each) and dried in vacuo. The peptide wascleaved from the resin as described earlier and the crude peptideproduct was analysed and purified as described below

[0288] HPLC Conditions

[0289] Gradient HPLC analysis was done using a Hewlett Packard HP 1100HPLC system consisting of a HP 1100 Quaternary Pump, a HP 1100Autosampler a HP 1100 Column Thermostat and HP 1100 Multiple

[0290] Wavelength Detector. Hewlett Packard Chemstation for LC software(rev. A.06.01) was used for instrument control and data acquisition. Thefollowing columns and HPLC buffer system was used:

[0291] Column: VYDAC 238TP5415, C-18, 5 mm, 300Å 150×4.6 mm.

[0292] Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90% MeCN.

[0293] Gradient:

[0294] 0-1.5 min. 0% B

[0295] 1.5-25 min 50% B

[0296] 25-30 min 100% B

[0297] 30-35 min 100% B

[0298] 35-40 min 0% B

[0299] Flow 1, ml/min, oven temperature 40° C., UV detection: I=215 nm.HPLC purification of the crude peptide

[0300] The crude peptide products were purified PerSeptive BiosystemsVISION Workstation. VISION 3.0 software was used for instrument controland data acquisition. The following column and HPLC buffer system wasused:

[0301] Column: Kromasil KR 100Å, 10 mm C-8, 250×50.8 mm.

[0302] Buffer system: Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10%MQV, 90% MeCN.

[0303] Gradient: 0-37 min. 0-40% B

[0304] Flow 35 ml/min, UV detection: I=215 nm and 280 nm.

[0305] Mass Spectroscopy

[0306] The peptides were dissolved in super gradient methanol (Labscan,Dublin, Ireland), milli-Q water (Millipore, Bedford, Mass.) and formicacid (Merck, Damstadt, Germany) (50:50:0.1 v/v/v) to give concentrationsbetween 1 and 10 mg/ml. The peptide solutions (20 ml) were analysed inpositive polarity mode by ESI-TOF-MS using a LCT mass spectrometer(Micromass, Manchester, UK).

[0307] Antibody Preparation And Use

[0308] Antibodies of the invention can be prepared by techniquesgenerally known in the field. Preferred antibodies are generated to apurified sample of peptide or peptide conjugate antigen. Suitablepeptide and peptide conjugate antigens for making such antibodies aredisclosed throughout the application including Examples 22-23, below.Although most polyclonal antibodies of the invention featureexceptionally good and specific binding, for some applications it may beas useful or more useful to employ monoclonal antibodies. See generallyHarlow, E and D. Lane in Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y. (disclosing methods for making and using polyclonal andmonoclonal antibodies).

[0309] Generally, antibodies can be prepared by immunizing a mammal witha purified or semi-purified sample of the peptide or peptide conjugateantigen as provided herein, alone or complexed with a carrier. Suitablemammals include typical laboratory animals such as sheep, goats,rabbits, guinea pigs, rats and mice. Rats and mice, especially mice, arepreferred for obtaining monoclonal antibodies. The antigen can beadministered to the mammal by any of a number of suitable routes such assubcutaneous, intraperitoneal, intravenous, intramuscular orintracutaneous injection. The optimal immunizing interval, immunizingdose, etc. can vary within relatively wide ranges and can be determinedempirically based on this disclosure. Typical procedures involveinjection of the antigen several times over a number of months.Antibodies are collected from serum of the immunized animal by standardtechniques and screened to find antibodies specific for the peptide orpeptide conjugate antigen used. Monoclonal antibodies can be produced incells which produce antibodies and those cells used to generatemonoclonal antibodies by using standard fusion techniques for forminghybridoma cells. See G. Kohler, et al., Nature, 256:456 (1975).Typically this involves fusing an antibody producing cell with animmortal cell line such as a myeloma cell to produce the hybrid cell.Alternatively, monoclonal antibodies can be produced from cells by themethod of Huse, et al., Science, 256:1275 (1989).

[0310] See also Harlow, E. and D. Lane, supra, for additionalinformation relating to making and using polyclonal and monoclonalantibodies. A variety of suitable antibody purification strategies arereported which can be used in accord with this invention.

[0311] In embodiments in which monoclonal antibodies are desired, onesuitable protocol provides for intraperitoneal immunization of a mousewith a composition comprising purified peptide conjugate conducted overa period of about two to seven months. Spleen cells then can be removedfrom the immunized mouse. Sera from the immunized mouse is assayed fortiters of antibodies specific for a particular peptide conjugate priorto excision of spleen cells. The excised mouse spleen cells are thenfused to an appropriate homogenic or heterogenic (preferably homogenic)lymphoid cell line having a marker such as hypoxanthine-guaninephosphoribosyltransferase deficiency (HGPRT) or thymidine kinasedeficiency (TK). Preferably a myeloma cell is employed as the lymphoidcell line. Myeloma cells and spleen cells are mixed together, e.g. at aratio of about 1 to 4 myeloma cells to spleen cells. The cells can befused by the polyethylene glycol (PEG) method. See G. Kohler, et al.,Nature, supra. The thus cloned hybridoma is grown in a culture medium,e.g. RPMI-1640. See G. E. More, et al., Journal of American MedicalAssociation, 199:549 (1967). Hybridomas, grown after the fusionprocedure, are screened such as by radioimmunoassay or enzymeimmunoassay for secretion of antibodies that bind specifically to theantigen employed e.g. antibodies are selected that bind to the purifiedpeptide conjugate but not to other unrelated control peptides.Preferably an ELISA or related immunological assay is employed for thescreen. Hybridomas that show positive results upon such screening can beexpanded and cloned by limiting dilution method. Further screens arepreferably performed to select antibodies that can bind to peptideconjugate in solution. The isolated antibodies can be further purifiedby nearly any suitable immunological technique including affinitychromatography.

[0312] The molecular weight of the antibodies of the invention will varydepending on several factors such as the intended use and whether theantibody includes a conjugated or recombinantly fused toxin,pharmaceutical, or detectable label or the like. In general, an antibodyof the invention will have a molecular weight of between approximately20 to 150 kDa. Such molecular weights can be readily are determined bymolecular sizing methods such as SDS-PAGE gel electrophoresis followedby protein staining or Western blot analysis.

[0313] Preferred peptide conjugates for making polyclonal antibodies ofthe invention are disclosed eg., in Examples 22-23.

[0314] By the phrase “specific binding” or related phrase as it relatesto association between an antibody and peptide or peptide conjugateantigen is meant that the antibody forms an immune complex with theparticular antigen and not with other antigens (such as related orunrelated peptide conjugates). Methods for detecting and optionallyquantifying such specific binding include standard immmunoassays eg.,ELISA, antibody capture and antigen capture assays. Preferred antibodiesof the invention specifically bind a subject peptide or peptideconjugate antigen. See Example 23 below, for instance.

[0315] The following examples are provided to point out preferredaspects of the invention and are not intended to be indicative of thescope of the invention.

SYNTHESIS EXAMPLES Example 1 Synthesis of Compound 1Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0316] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 390mg. After purification using preparative HPLC as earlier described, 210mg peptide product was collected with a purity better than 95% and theidentity of the peptide was confirmed by MS (found M 1780.25, calculatedM 1780.11).

Example 2 Synthesis of Compound 1AAc-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂×9 AcOH (acetatesalt). Counter Ion Exchange from Trifluoroacetate to Acetate of Compound1

[0317] The purified synthetic peptide product of compound 1 is isolatedas a trifluoroacetate salt,Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2×9 TFA, due to thepresence of trifluoroacetic acid (0.1% v/v) in the HPLC buffers used forthe purification of the crude synthetic peptide product. In order toexchange the counter ion trifluoroacetate with acetate, a solution ofthe peptide was passed through a column packed with strong base ionexchange resin on the acetate (Dowex 1×8). 1 g Compound 1 is dissolvedin 40 ml water. The solution is passed through a column containing 40 mlstrong base ion exchange resin on the acetate (Dowex 1×8; capacity 1.33meq/ml resin). The resin is then washed with 4×30 ml water and theeluate is collected and lyophilized resulting in 792 mg acetate salt(hygroscopic) with a purity according to HPLC analysis of 98%.

Example 3 Synthesis of Compound 1CAc-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂9 HCl (ChlorideSalt). Counter Ion Exchange from Trifluoroacetate (Tfa) to Chloride(Cl—) of Compound 1

[0318] 100 mg Compound 1 was dissolved in 50 ml 0.1M hydrochloric acidand the resulting solution was lyophilized. The remanence was dissolvedin 50 ml water and lyophilized again resulting in 74 mg of the chloridesalt with a purity according to HPLC of 97%.

Example 4 Synthesis of Compound 2Ac-Lys-Lys-Lys-Lys-Lys-Lys-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0319] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalLysine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 231mg. After purification using preparative HPLC as described above, 126 mgpeptide product was collected with a purity better than 93% and theidentity of the peptide was confirmed by MS (found M 1780.25, calculatedM 1780.11).

Example 5 Synthesis of Compound 3H-Asn-Glu-Glu-Glu-Glu-Glu-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂on TentaGel S RAM-Lys(Boc)Fmoc

[0320] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalAsparagine. All couplings were continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 212mg. After purification using preparative HPLC as described above, 112 mgpeptide product was collected with a purity better than 98% and theidentity of the peptide was confirmed by MS (found M 2497.25 calculatedM 2497.35).

Example 6 Synthesis of Compound 4Ac-Arg-Tyr-Asn-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0321] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 502mg. After purification using preparative HPLC as earlier described, 272mg peptide product was collected with a purity better than 98% and theidentity of the peptide was confirmed by MS (found M 1731.25, calculatedM 1731.09).

Example 7 Synthesis of Compound 5Ac-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Trp-Arg-Tyr-Tyr-Asn-NH₂ on TentaGel S RAM

[0322] Dry TentaGel S RAM (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Lysine. Allcouplings were continued over night. After deprotection of the Fmocgroup the N-terminal amino group was acetylated as described above. Thecoupling was continued over night. The acylations were checked asearlier described. After completed synthesis the peptide was cleavedfrom the resin as described above. Yield of crude product 586 mg. Afterpurification using preparative HPLC as earlier described, 365 mg peptideproduct was collected with a purity better than 99% and the identity ofthe peptide was confirmed by MS (found M 1738.0, calculated M 1738.05).

Example 8 Synthesis of Compound 6Ac-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Trp-Arg-Tyr-Tyr-Arg-NH₂ on TentaGel S RAM

[0323] Dry TentaGel S RAM (0.23 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalLysine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 437mg. After purification using preparative HPLC as earlier described, 257mg peptide product was collected with a purity better than 98% and theidentity of the peptide was confirmed by MS (found M 1780.0, calculatedM 1780.11).

Example 9 Synthesis of Compound 7Ac-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Trp-D-Arg-D-Tyr-D-Tyr-D-Arg-NH₂on TentaGel S RAM

[0324] Dry TentaGel S RAM (0.23 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalD-Lysine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 410mg. After purification using preparative HPLC as earlier described, 263mg peptide product was collected with a purity better than 98% and theidentity of the peptide was confirmed by MS (found M 1780.13, calculatedM 1780.11).

Example 10 Synthesis of Compound 8Ac-D-Arg-D-Tyr-D-Tyr-D-Arg-D-Trp-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-NH₂on TentaGel S RAM

[0325] Dry TentaGel S RAM (0.23 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalD-Arginine. All couplings were continued over night. After deprotectionof the Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 419mg. After purification using preparative HPLC as earlier described, 205mg peptide product was collected with a purity better than 97% and theidentity of the peptide was confirmed by MS (found M 1780.0, calculatedM 1780.11).

Example 11 Synthesis of Compound 9Ac-Lys-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0326] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalLysine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 445mg. After purification using preparative HPLC as earlier described, 287mg peptide product was collected with a purity better than 96% and theidentity of the peptide was confirmed by MS (found M 1752.0, calculatedM 1752.1).

Example 12 Synthesis of Compound 10Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0327] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 376mg. After purification using preparative HPLC as earlier described, 134mg peptide product was collected with a purity better than 97% and theidentity of the peptide was confirmed by MS (found M 1707.0, calculatedM 1707.11).

Example 13 Synthesis of Compound 11Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Ala-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0328] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 429mg. After purification using preparative HPLC as earlier described, 260mg peptide product was collected with a purity better than 96% and theidentity of the peptide was confirmed by MS (found M 1723.0, calculatedM 1723.05).

Example 14 Synthesis of Compound 12Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0329] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 426mg. After purification using preparative HPLC as earlier described, 266mg peptide product was collected with a purity better than 98% and theidentity of the peptide was confirmed by MS (found M 1651.88, calculatedM 1652.01).

Example 15 Synthesis of Compound 13Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Cys-NH₂ on TentaGel SRAM

[0330] Dry TentaGel S RAM (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 465mg. After purification using preparative HPLC as earlier described, 313mg peptide product was collected with a purity better than 91% and theidentity of the peptide was confirmed by MS (found M 1883.0, calculatedM 1883.12).

Example 16 Synthesis of Compound 15 Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Cys-NH₂on TentaGel S RAM

[0331] Dry TentaGel S RAM (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Arginine. Allcouplings were continued over night. After deprotection of the Fmocgroup the N-terminal amino group was acetylated as described above. Thecoupling was continued over night. The acylations were checked asearlier described. After completed synthesis the peptide was cleavedfrom the resin as described above. Yield of crude product 249 mg. Afterpurification using preparative HPLC as earlier described, 190 mg peptideproduct was collected with a purity better than 94% and the identity ofthe peptide was confirmed by MS (found M 1114.50, calculated M 1114.55).

Example 17 Synthesis of Compound 16H-Cys-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0332] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalCysteine. All couplings were continued over night. The acylations werechecked as earlier described. After the final deprotection of the Fmocgroup the peptide was cleaved from the resin as described above. Yieldof crude product 359 mg. After purification using preparative HPLC asearlier described, 242 mg peptide product was collected with a puritybetter than 98% and the identity of the peptide was confirmed by MS(found M 1240.76, calculated M 1240.78).

Example 18 Synthesis of Compound 17 H-Cys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ onTentaGel S RAM-Lys(Boc)Fmoc

[0333] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalCysteine. All couplings were continued over night. The acylations werechecked as earlier described. After the final deprotection of the Fmocgroup the peptide was cleaved from the resin as described above. Yieldof crude product 287 mg. After purification using preparative HPLC asearlier described, 205 mg peptide product was collected with a puritybetter than 90% and the identity of the peptide was confirmed by MS(found M 888.5, calculated M 888.61).

Example 19 Synthesis of Compound 18Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Cys-NH₂ on TentaGel SRAM

[0334] Dry TentaGel S RAM (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Arginine. Allcouplings were continued over night After deprotection of the Fmoc groupthe N-terminal amino group was acetylated as described above. Thecoupling was continued over night. The acylations were checked asearlier described. After completed synthesis the peptide was cleavedfrom the resin as described above. Yield of crude product 465 mg. Afterpurification using preparative HPLC as earlier described, 313 mg peptideproduct was collected with a purity better than 91% and the identity ofthe peptide was confirmed by MS (found M 1883.00, calculated M 1883.12).

Example 20 Synthesis of Compound 14Tfa-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ on TentaGel SRAM-Lys(Boc)Fmoc

[0335] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration.and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was trifluoroacetylated asdescribed above. The reaction was continued over night. The acylationswere checked as earlier described. After completed synthesis the peptidewas cleaved from the resin as described above. Yield of crude product392 mg. After purification using preparative HPLC as earlier described,105 mg peptide product was collected with a purity better than 96% andthe identity of the peptide was confirmed by MS (found M 1834.25,calculated M 1834.08).

Example 21 Synthesis of Compound CE1 Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ onTentaGel S RAM-Lys(Boc)Fmoc

[0336] Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed ina polyethylene vessel equipped with a polypropylene filter forfiltration and treated as described under “batchwise peptide synthesison TentaGel resin” until finishing the coupling of the N-terminalArginine. All couplings were continued over night. After deprotection ofthe Fmoc group the N-terminal amino group was acetylated as describedabove. The coupling was continued over night. The acylations werechecked as earlier described. After completed synthesis the peptide wascleaved from the resin as described above. Yield of crude product 143mg. After purification using preparative HPLC as earlier described, 52mg peptide product was collected with a purity better than 97% and theidentity of the peptide was confirmed by MS (found M 1011.54, calculatedM 1011.54).

Example 22 Immunogen Production

[0337] Antibodies specific for Compound 1 have been raised byconjugating peptides corresponding to the N-terminus (Ac-RYYRWKC-NH₂),the C-terminus (H-CAPPSKKKKKK-NH₂) and Compound 13 which is a fulllength molecule having a terminal Cys residue to accommodate thecoupling chemistry (Ac-RYYRWKKKKKKKC-NH₂). Ac-RYYRWKC-NH₂ andH-CAPPSKKKKKK-NH₂ were coupled to keyhole limpet hemocyanin (KLH) and acommercially available cationised bovine serum albumin (Supercarrier,Pierce Chemical). The resulting KLH and Supercarrier conjugates werecombined, and injected into rabbits. Compound 13 was conjugated topurified protein derivative (PPD) and injected into a BCG primed goat.Immunisations followed standard protocols of initial immunisation ofimmunogen in an emulsion of Freund's complete adjuvant followed by aregimen of boosting with an emulsion of the immunogen in Freund'sincomplete adjuvant and test bleeding until antibody titers were atacceptable levels. The antibodies were purified in one step by affinitychromatography on a protein G column specific for the IgG subclass ofimmunoglobulins.

Example 23 Antibody Production

[0338] Antibodies were prepared generally along lines discussedpreviously. Four representative polyclonal antibodies have been raisedto peptides fitting the general formulas described in the claims. Thesecomprise a peptide incorporating the sequence of N-terminus of compound1, two peptides incorporating the sequence of the C-terminus of compound1, as well as a peptide incorporating the full sequence of compound 1.These peptides are designated compounds 15, 16, 17, or 18, respectively,and have the following sequences: Ac-RYYRWKKKKKKKC-NH₂ (compound 15);CAPPSKKKKKK-NH₂ (compound 16); CKKKKKK-NH₂ (compound 17) andAc-RYYRWKKKKKKKC-NH₂ (compound 18). Polyclonal antibodies to compounds15, 16, and 17 were raised in rabbits, and to compound 18 in goat. Thesepolyclonal antibodies are highly specific and result in titers listed inthe accompanying table when the antibodies are evaluated in a standardELISA type assay employing Compound I as the antigen and utilizingcalorimetric detection of bound antibody by a commercially availableHRP-conjugated anti-lgG antibody. Preliminary testing for several of theantibodies has also indicated the lack of cross reaction with antigensof low sequence similarity to compound 1 in the standard ELISA assaydescribed above. Table 7 is shown below. TABLE 7 Immunogen Antigen inELISA Antibody titer 15 Compound 1 1:750,000 16 Compound 1 1:200,000 17Compound 1 1:350,000 17 Glucagon No Reaction 18 Compound 1 1:2000

[0339] See the Examples and discussion above for more informationrelating to the peptide conjugate compounds disclosed in this Example.

[0340] All references disclosed in this application are incorporatedherein by reference.

[0341] While the invention has been described with reference to specificembodiments, modifications and variations of the invention may beconstructed without departing from the scope of the invention, which isdefined in the following claims.

What is claimed is:
 1. A peptide conjugate of the general formula IR₁-Z-X-Z′-R₂  (I) wherein X represents a hexapeptide of the formula IIA¹-A²-A³-A⁴-A⁵-A⁶  (II) wherein A¹ represents Arg, Lys, or His, A²represents Tyr, Trp, or Phe, A³ represents Tyr, Asn, Trp or Phe, A⁴represents Lys, Arg or His, A⁵ represents Phe, Tyr, Trp, Leu, Val orlie, and A⁶ represents Arg, Lys or His and wherein each amino acidresidue in said hexapeptide may be in the L or D form; Z represents acharged peptide chain of from 4 to 20 amino acid residues having the Dor L configuration or is missing; and Z′ represents a charged peptidechain of from 4 to 20 amino acid residues having the D or Lconfiguration or is missing, providing that not both of Z and Z′ aremissing; R₁ represents H or an acyl group; R₂ represents NR₃R₄ whereeach of R₃ and R₄ independently represents hydrogen, C(1-6)alkoxy,aryloxy or a lower alkyl as defined herein; or R₂ represents OH; thepeptide conjugates of formula I being optionally further linked to atransport moiety; and salts, hydrates and solvates thereof, andC-terminally amidated or esterified derivatives thereof with suitableorganic or inorganic acids.
 2. A peptide conjugate according to claim 1wherein formula II is represented by the amino acid sequence(RK)YY(RK)(WI)(RK) wherein alternative amino acid residues at positions1, 4, 5 and 6 are shown in brackets.
 3. A peptide conjugate according toany one of the preceding claims wherein the number of amino acidresidues in Z and Z′ is in the range 5 to
 10. 4. A peptide conjugateaccording to any one of the preceding claims wherein the amino acidresidues in Z and Z′ have the L-configuration.
 5. A peptide conjugateaccording to any one of the preceding claims wherein the amino acidresidues of Z are selected from the group consisting of Q, T, S, P, N,E, and D.
 6. A peptide conjugate according to any one of the precedingclaim wherein the N-terminal amino acid of Z is selected from the groupconsisting of Q, T, N and S and the remaining amino acid residues areselected from the group consisting of P, D, and E.
 7. A peptideconjugate according to any one of the preceding claims wherein Z isselected from the group consisting of N(E)₇, N(E)₆, N(E)₅, N(E)₃, S(E)₇,S(E)₆, S(E)₅, S(E)₃, NP(E)₄, NP(E)₅, N(D)₇, N(D)₆, N(D)₅, N(D)₃, Q(E)₇,Q(E)₅, Q(E)₃, QN(D)₇, Q(D)₆, Q(D)₅, and Q(D)₃.
 8. A peptide conjugatehaving the general formula III R₁-X-Z′-R₂  (III) wherein R₁, X, Z′ andR₂ have the same meanings as defined in the preceding claims; and salts,hydrates and solvates thereof, and C-terminally amidated or esterifiedderivatives thereof with suitable organic or inorganic acids.
 9. Apeptide conjugate according to any one of the preceding claims whereinthe peptide sequence is in the all-D form, the retro form, or the retroall-D form.
 10. A peptide conjugate according to any one of thepreceding claims being optionally further linked to a transport moiety,where said transport moiety is preferably selected from the groupconsisting of a HIV tat peptide residues 49-57, HIV tat peptide residues49-56, the tat sequence YGRKKRRQRRR, a polyarginine peptide having from6 to 20 residues, such as R₆ , and transducing peptide sequences, suchas the following peptide sequences: YARKARRQARR, YARAAARQARA,YARAARRAARR, YARMRRAARA, YARRRRRRRRR, and YAAARRRRRRR.
 11. A peptideconjugate according to any one of the preceding claims wherein Z′represents a positively charged peptide chain.
 12. A peptide conjugateaccording to any one of the preceding claims wherein R₁ representsacetyl or trifluoroacetyl.
 13. A peptide conjugate according to any oneof the preceding claims wherein R₂ represents NH₂, or R₂ representsNR₃R₄ where each of R₃ and R₄ independently represents hydrogen, methylor ethyl.
 14. A peptide conjugate according to any one of the precedingclaims wherein said hexapeptide X is selected form the group consistingof KYYRWK, RYYRWR, RYYRWK, RYYRWK (all-D), KWRYYR, RYYRIK, RYYRIR,RYYKIK, RYYKIR, RYYKWR, and RYYKWK.
 15. A peptide conjugate according toany one of the preceding claims wherein said hexapeptide X is KYYRWK orRYYRWK.
 16. A peptide conjugate according to any one of the precedingclaims wherein the amino acid residues of Z′ are selected from the groupconsisting of A, G, K, H and R, preferably K.
 17. A peptide conjugateaccording to the preceding claim wherein Z′ is selected from the groupconsisting of A(K₄)G, K₅G, AK₅, K₁₀, K₉, K₈, K₇, K₆, K₅, K₄.
 18. Apeptide conjugate according to any one of the preceding claims furthercomprising a terminal cysteinyl residue.
 19. A peptide conjugateselected from the group consisting of Compound 1 Ac-RYYRWKKKKKKK-NH₂Compound 2 Ac-KKKKKKRYYRWK-NH₂ Compound 3 H-NEEEEERYYRWKKKKKKK-NH₂Compound 4 Ac-RYNRWKKKKKKK-NH₂ Compound 5 Ac-KKKKKKKWRYYN-NH₂ Compound 6Ac-KKKKKKKWRYYR-NH₂ Compound 7 Ac-KKKKKKKWRYYR-NH₂ (all D) Compound 8Ac-RYYRWKKKKKKK-NH₂ (all D) Compound 9 Ac-KYYRWKKKKKKK-NH₂ Compound 10Ac-RYYRIKKKKKKK-NH₂ Compound 11 Ac-RYYRWKAKKKKK-NH₂ Compound 12Ac-RYYRWKKKKKK-NH₂ Compound 13 Ac-RYYRWKKKKKKKC-NH₂ Compound 14Tfa-RYYRWKKKKKKK-NH₂

the C-terminal free acid thereof, esterified derivatives thereof, andpharmaceutically acceptable acid addition salts thereof.
 20. A peptideconjugate according to claim 19 which is Compound 1AAc-RYYRWKKKKKKK-NH₂×9CH₃COOH or Compound 1C Ac-RYYRWKKKKKKK-NH₂×9HCl 21.A peptide conjugate X-Z′ selected from the group consisting ofRYYRWKAK₅, KYYRWKK₆, RYYRWKK₆, RYYRWRK₆, RYYRIKK₆, RYYRWK₅ andpharmaceutically acceptable salts and derivatives thereof includingN-terminally acetylated and C-terminally amidated or esterifiedderivatives.
 22. A peptide conjugate according to formula I or III whichis Ac-RYYRWKK₆-NH₂(Compound 1) and salts, hydrates, solvates andN-terminally acylated derivatives or esters thereof.
 23. A biologicallyactive substance comprising a peptide conjugate of formula I or IIIwhich is positively charged and a negatively charged counterion selectedfrom the group consisting of pharmaceutically acceptable anions,preferably CH₃COO⁻, CF₃COO⁻, Cl⁻, SO₃ ²⁻, maleate and oleate.
 24. Apharmaceutical composition comprising as the active compound a peptideconjugate of formula I or III or an active substance as defined in claim23 and a pharmaceutically acceptable carrier or diluent.
 25. Apharmaceutical composition according to claim 24 which further comprisesa liquid carrier selected from the group consisting of syrup, peanutoil, olive oil, phospholipids, fatty acids, fatty acid amines,polyoxyethylene and water for parenteral administration.
 26. Apharmaceutical composition according to the preceding claim containingin a unit dosage an amount of a compound of formula I or III rangingfrom about 0.1 to about 10 mg.
 27. A pharmaceutical compositioncomprising as an active compound a peptide conjugate of formula I or IIIor an active substance as defined in claim 23 in a form adapted forperipheral administration only.
 28. Use of a peptide conjugate offormula I or III or an active substance as defined in claim 23 for thepreparation of a medicament.
 29. Use of a peptide conjugate of formula Ior III or an active substance as defined in claim 23 for the preparationof a medicament for the treatment and/or prevention of hyponatremia. 30.Use according to the preceding claim wherein said hyponatremia isassociated with heart failure.
 31. Use according to claim 30 whereinsaid hyponatremia is associated with intensive diuretic therapy withthiazides and/or loop diuretics.
 32. Use of a peptide conjugate offormula I or III or an active substance as defined in claim 23 for thepreparation of a medicament for selective water diuresis.
 33. Use of apeptide conjugate of formula I or III or an active substance as definedin claim 23 for the preparation of a medicament for the treatment and/orprevention of a water retaining condition, such as congestive heartfailure, liver cirrhosis, nephrotic syndrome and hypertension.
 34. Useof a peptide conjugate of formula I or III or an active substance asdefined in claim 23 for the preparation of a medicament for thetreatment and/or prevention of multiple organ failure.
 35. Use of apeptide conjugate of formula I or III or an active substance as definedin claim 23 for the preparation of a medicament for the treatment and/orprevention of acute renal failure.
 36. Use of a peptide conjugate offormula I or III or an active substance as defined in claim 23 for thepreparation of a medicament to be used in the treatment of diseasestates associated with elevated tone of nociceptin.
 37. Use of a peptideconjugate of formula I or III or an active substance as defined in claim23 for the preparation of a medicament for the treatment of hypokalemia.38. Use according to the preceding claim wherein said hypokalemia isassociated with intensive diuretic therapy with thiazides and/or loopdiuretics.
 39. Use of a peptide conjugate of formula I or III or anactive substance as defined in claim 23 for the preparation of amedicament for the treatment of hypertension.
 40. A method of treatingor preventing hyponatremia or hypokalemia comprising administering atherapeutically effective amount of a peptide conjugate of formula I orIII or an active substance as defined in claim 23 to a patient in needthereof.
 41. A method of treating or preventing a water retainingcondition, such as congestive heart failure, liver cirrhosis, nephroticsyndrome and hypertension, comprising administering a therapeuticallyeffective amount of a peptide conjugate of formula I or III or an activesubstance as defined in claim 23 to a patient in need thereof.
 42. Amethod of treating or preventing acute renal failure comprisingadministering a therapeutically effective amount of a peptide conjugateof formula I or III or an active substance as defined in claim 23 topatient in need thereof.
 43. A method of treating or preventing multipleorgan failure comprising administering a therapeutically effectiveamount of a peptide conjugate of formula I or III or an active substanceas defined in claim 23 to patient in need thereof.
 44. A method oftreating or preventing hypertension comprising administering atherapeutically effective amount of a peptide conjugate of formula I orIII or an active substance as defined in claim 23 to a patient in needthereof.
 45. A method of treating edema comprising administering to apatient in need of such treatment a therapeutically effective amount ofa peptide conjugate of formula I or III or an active substance asdefined in claim 23 optionally in combination with a diuretic.
 46. Themethod of claim 45, wherein the edema is associated with coronary heartfailure.
 47. The method according to claim 43 wherein said diuretic is aloop diuretic.
 48. A nucleic acid sequence encoding a polypeptidesequence comprising the peptide sequence of formula I or III.
 49. Arecombinant host cell comprising the nucleic acid sequence of claim 48and capable of expressing said polypeptide sequence.
 50. A method forproducing the peptide conjugate of formula I or III having a naturalpolypeptide sequence, comprising introducing a nucleic acid sequenceencoding a polypeptide sequence comprising the peptide sequence offormula I or III and a selectable marker contained within a nucleic acidconstruct or a vector into a host cell to obtain a recombinant hostcell; selecting said recombinant host cell; culturing said recombinanthost cells under conditions permitting the production of saidpolypeptide sequence; isolating said polypeptide sequence from theculture; and optionally cleaving said polypeptide sequence using anappropriate protease to obtain said peptide conjugate.
 51. A method ofsynthesising the peptide conjugate of the invention according tosolution phase and solid phase techniques as described herein.
 52. Amethod of enhancing the stability of a hexapeptide in blood plasma,preferably a hexapeptide of formula II herein by linking a peptidesequence Z′ as defined herein, via a peptide bond to the C-terminus ofsaid hexapeptide to form a conjugated peptide, preferably of formula IIIherein.
 53. A method according to claim 52 wherein said hexapeptide isselected from the group of hexapeptides defined by the aminoacidsequence (RK)YY(RK)(WI)(RK) wherein alternative amino acid residues atpositions 1, 4, 5 and 6 are shown in brackets.
 54. A method according toany one of claims 52 and 53 wherein said sequence Z′ is selected fromthe group consisting of unbranched K₅, K₆ and K₅C sequences.
 55. Use ofa peptide conjugate of formula I or III or an epitopic fragment thereofcomprising a part or all of the X sequence of formula II or the formula(RK)YY(RK)(WI)(RK) and/or a part or all of the Z or Z′ sequencespreferably coupled to a carrier through a terminal cysteinyl residue forraising antibodies capable of specifically binding to said peptideconjugate or said X sequence.
 56. Use of Compound 15Ac-RYYRWKKKKKKKC-NH₂ for raising an antibody capable of specificallybinding to a peptide conjugate of formula I or III or a fraction of thepeptide sequence thereof.
 57. An antibody that specifically binds one ofthe peptide conjugates represented by formula I and III.
 58. Theantibody of claim 57, wherein the antibody has an IgG serotype.
 59. Theantibody of claim 57 or 58, wherein the antibody is polyclonal ormonoclonal.
 60. The antibody of claim 59, wherein the antibody ispolyclonal and specifically binds one of the following peptides:Ac-RYYRWKKKKKKKC-NH₂ (Compound 15); CAPPSKKKKKK-NH₂ (Compound 16);CKKKKKK-NH₂ (Compound 17), and Ac-RYYRWKKKKKKKC-NH₂ (Compound 18). 61.Use of a peptide conjugate of formula I or III or an epitopic fragmentthereof comprising a part or all of the X sequence of formula II or theformula (RK)YY(RK)(WI)(RK) and/or a part or all of the Z or Z′ sequencespreferably coupled to a carrier through a terminal cysteinyl residue forraising antibodies capable of specifically binding to said peptideconjugate or said X sequence.
 62. Use of a peptide selected from thegroup consisting of Compound 15 Ac-RYYRWKKKKKKKC-NH₂, Ac-RYYRWKC-NH₂,CKKKKKK (Compound 17) and H-CAPPSKKKKKK-NH₂, for raising an antibodycapable of specifically binding to peptide conjugates of formula I orIII or a fragment of the peptide sequence thereof.
 63. A specific IgGantibody against the peptide conjugates of formula I and III produced asdescribed herein.